From novalidaddress from nurfuerspam.de Thu Jan 1 08:44:21 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Jan 1 12:01:23 2009 Subject: Make Akta Prime Software work under Linux!? Message-ID: <00c6ebc5-1d53-4bb3-9873-ae68c997bb3f@r24g2000vbp.googlegroups.com> Dear Colleagues, I am about to make the ?kta Prime package run with Opensuse Linux 11.1 (WINE 1.1.9, Kernel 2.6.27.7-9-default). I already successfully have set up the Prime software to acquire data from the machine (for monitoring runs, "Csyscon.exe prime"). However, I have serious problems doing the same with the Evaluation software ("Emain.exe prime") that is used to analyze the data. Currently, I am figthing with DLLs (MFC42.DLL and MSVCRT.DLL in detail) as I get error messages pointing to unresolved functions, the app crashed after loading any data set. I already have tried all versions of these DLLs I could get hold of, but no success yet. Has anyone already solved the "problem" or wants to do the same and is willing to share her/his experience? Or is interested to team up? Best regards, Wolfgang (?kta/Akta Prime is an FPLC protein purification machine from Pharmacia/Amersham/GE-Healthcare) From amlee3 from ncsu.edu Thu Jan 1 14:09:30 2009 From: amlee3 from ncsu.edu (amlee3@ncsu.edu) Date: Thu Jan 1 14:48:29 2009 Subject: SOD assay questions Message-ID: <2201.152.1.213.16.1230836970.squirrel@webmail.ncsu.edu> Hi, I was wondering if anyone knows of a good way to prepare xanthine oxidase (bought Xanthine Oxidase from bovine milk, 25Units, Sigma Aldrich Cat. no. X4500) for an SOD assay using the reduction of Cytochrome C method? ( McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055) I'd like to maximize the activity of xanthine oxidase and its longevity. Several suggestions from other labs said i needed to make it fresh each time, but that seems like a waste, others suggest resuspension in a wing buffer (50mM Pot phosphate, 0.1mM EDTA pH7.8) then dialyze it overnight. Any ideas? A detailed protocol would be really helpful. Thanks so much. Alice From novalidaddress from nurfuerspam.de Thu Jan 1 15:05:44 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Thu Jan 1 23:32:03 2009 Subject: SOD assay questions References: Message-ID: <3e82e4f5-8e1f-4927-b813-861d1c1a72af@40g2000prx.googlegroups.com> Hi Alice, you could add 1M (final) hydroxyectoine (e.g from http://www.bitop.de or from Fluka by http://www.sial.com - I am aware of these 2 sources) as stabilizer. Then do not freeze but store aliquots in the fridge. If you need to filter sterilize, use regenerated cellulose (RC) syringe filters, other might refuse to be permeable (ectoines are sort of funny (???) compunds...). If possible, dissolve the enzyme in the prepared buffer or alternatively dissolve in 2x buffer and add the same volume of 2M hydroxyectoine. Ectoine (cheaper, but also a compatible solute) might work as well. Another option (without (hydroxy)ectoine) might be dispensing aliquots into eppis and snap-freeze them in liquid nitrogen. Then store frozen, preferably at -80degC or colder. Best regards, Wo From Paul.Phelan from tufts.edu Thu Jan 1 18:31:05 2009 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Thu Jan 1 23:32:12 2009 Subject: Electrophoresis of dsDNA and ssDNA Message-ID: <20090101183105.kbzke9oge80oo44c@webmail.tufts.edu> I have a simple question that so far does not seem to have a simple answer. I need to confirm that a 60-mer dsDNA oligo is double-stranded. This is poly-dA/dT DNA. Does anyone have a straightforward 1D electrophoresis protocol that will separate single-stranded and double-stranded forms of an oligonucleotide in the same range as a 60-mer oligo? I have already been surprised to learn that in 7 M urea PAGE, ssDNA will run SLOWER than dsDNA. Does anyone have experience with this? Thanks in advance, Paul From glenn.dunshea from gmail.com Fri Jan 2 02:25:59 2009 From: glenn.dunshea from gmail.com (Glenn Dunshea) Date: Fri Jan 2 13:10:25 2009 Subject: Electrophoresis of dsDNA and ssDNA In-Reply-To: <20090101183105.kbzke9oge80oo44c@webmail.tufts.edu> References: <20090101183105.kbzke9oge80oo44c@webmail.tufts.edu> Message-ID: <8acca5110901012325p1d719731t879471e1fad6e2d8@mail.gmail.com> I do SSCP analysis often and can confirm that with PCR product around 200bp, the single stranded DNA runs much slower then double stranded. (I can send you a pic if you like). Here is a ballpark suggestion you may wish to try: You may want to try denaturing the sample you wish to confirm is dsDNA and see if it produces the same electrophoretic banding as a non denatured sample. Heat an aliquot at 95 degree's for a few minutes, take off and add one volume of stop solution (95% formamide, 10mM NaOH, 0.25% bromo blue, 0.25% xylene cyanol). Run this out in a PAGE gel (I am unsure of an appropriate PAGE mix suitable for SSCP type things - I just use 1x pre-mixed MDE solution but I'm sure you could find an appropriate PAGE mix in the literature somewhere) beside another aliquot of the same not denatured. If they produce the same electrophoretic band pattern then the sample is single stranded. If it is double stranded there will be additional bands closer to the well in the denatured sample. Short of a few details I know but potentially a start? On Fri, Jan 2, 2009 at 8:31 AM, Paul J. Phelan wrote: > I have a simple question that so far does not seem to have a simple answer. > I need to confirm that a 60-mer dsDNA oligo is double-stranded. > This is poly-dA/dT DNA. Does anyone have a straightforward 1D > electrophoresis protocol that will separate single-stranded and > double-stranded forms of an oligonucleotide in the same range as a 60-mer > oligo? > I have already been surprised to learn that in 7 M urea PAGE, ssDNA will > run SLOWER than dsDNA. Does anyone have experience with this? > > Thanks in advance, > > Paul > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From vedha.kv from gmail.com Fri Jan 2 05:27:49 2009 From: vedha.kv from gmail.com (vedha giri) Date: Fri Jan 2 13:10:32 2009 Subject: Clarification for LacZ gene in expression vectors Message-ID: Dear Sir, If I Ligate my gene of interest in to the expression vector that do not have the LacZ gene, what will be result of Blue /white selection test? How should I conformed my insert result? Regards -- K.Vedhagiri Research Scholar, Dr K.Natarajseenivasan Lab, Department of Microbiology, Bharathidasan University, Tiruchirappalli - 620 024. India. Mobile +91 99446-83728 From bmacgreg from unc.edu Fri Jan 2 13:58:32 2009 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Fri Jan 2 15:57:07 2009 Subject: dsDNA and ssDNA In-Reply-To: <200901021703.n02H3Nj15800@net.bio.net> References: <200901021703.n02H3Nj15800@net.bio.net> Message-ID: > > > From: "Paul J. Phelan" > Date: January 1, 2009 6:31:05 PM EST > To: methods@magpie.bio.indiana.edu > Subject: Electrophoresis of dsDNA and ssDNA > > > I have a simple question that so far does not seem to have a simple > answer. > I need to confirm that a 60-mer dsDNA oligo is double-stranded. > This is poly-dA/dT DNA. Does anyone have a straightforward 1D > electrophoresis protocol that will separate single-stranded and > double-stranded forms of an oligonucleotide in the same range as a > 60-mer oligo? > I have already been surprised to learn that in 7 M urea PAGE, ssDNA > will run SLOWER than dsDNA. Does anyone have experience with this? > > Thanks in advance, > > Paul > Hi, You might try digestion with enzymes specific for ss or ds DNA - for example, E. coli Exo I is specific for ssDNA. Somebody should be able to spare a little... That's probably the simplest way. If you have the ss oligos, you could run them side by side with the putative ds oligo on e.g. the 7M urea PAGE gel, hopefully things would line up nicely. You could also try running the sample with and without heat denaturation, to see if extra bands appear, but that might not give an unambiguous result. As a control I suppose you could try generating restriction fragments of the same approximate size from some convenient DNA, run them with and without heat treatment - although it would probably take somewhat higher temperature/longer incubation to denature DNA in general than your all-AT oligo. Okay, end of random ideas, maybe someone has an easier answer. Barbara MacGregor From ddloeb from gmail.com Fri Jan 2 11:50:40 2009 From: ddloeb from gmail.com (ddloeb@gmail.com) Date: Fri Jan 2 15:57:20 2009 Subject: Electrophoresis of dsDNA and ssDNA References: Message-ID: <9945ea8f-187c-4f1e-a046-3cb856ed50cf@r10g2000prf.googlegroups.com> On Jan 1, 5:31?pm, "Paul J. Phelan" wrote: > I have a simple question that so far does not seem to have a simple answer. > I need to confirm that a 60-mer dsDNA oligo is double-stranded. > This is poly-dA/dT DNA. ?Does anyone have a straightforward 1D > electrophoresis protocol that will separate single-stranded and > double-stranded forms of an oligonucleotide in the same range as a > 60-mer oligo? Run them out on a 10% native PAGE gel in 1XTBE. Run the gel at 17oC. This procedure was used to separate fully-folded tRNA from partially unfolded forms. > I have already been surprised to learn that in 7 M urea PAGE, ssDNA > will run SLOWER than dsDNA. ?Does anyone have experience with this? In 7M urea, it is extremely unlikely that you have any dsDNA - especially for something that is AT-rich. Dan From ddloeb from gmail.com Fri Jan 2 12:45:29 2009 From: ddloeb from gmail.com (ddloeb@gmail.com) Date: Fri Jan 2 15:57:25 2009 Subject: Make Akta Prime Software work under Linux!? References: Message-ID: On Jan 1, 7:33?am, WS wrote: > Dear Colleagues, > > I am about to make the ?kta Prime package run with Opensuse Linux 11.1 > (WINE 1.1.9, Kernel 2.6.27.7-9-default). I already successfully have > set up the Prime software to acquire data from the machine (for > monitoring runs, "Csyscon.exe prime"). However, I have serious > problems doing the same with the Evaluation software ("Emain.exe > prime") that is used to analyze the data. Currently, I am figthing > with DLLs (MFC42.DLL and MSVCRT.DLL in detail) as I get error messages > pointing to unresolved functions, the app crashed after loading any > data set. I already have tried all versions of these DLLs I could get > hold of, but no success yet. I don't use Akta Prime (or Wine), but in general I have three suggestions: a) Have you tried Codeweavers Crossover-Linux? It is based on Wine, but is compatible with more Windows applications than Wine is. b) I use VMWare - haven't found a single application that doesn't work with it yet. But then again, they're running in a Windows XP guest session running on Linux. c) You could try Bochs. It is a little slower than VMWare - differences in virtualization routines, but Bochs is free Dan From gerchman from research.haifa.ac.il Sat Jan 3 11:38:24 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Sat Jan 3 13:55:37 2009 Subject: Heto FD3 freeze dryer manual Message-ID: <1231000704.495f948044ed3@webmail.haifa.ac.il> Greetings I am looking for Heto FD3 freeze dryer manual. Anyone willing to scan the important parts and sent? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From novalidaddress from nurfuerspam.de Sat Jan 3 05:46:14 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Jan 3 15:56:01 2009 Subject: Make Akta Prime Software work under Linux!? References: Message-ID: Hi Dan, I just tried your suggestion with the Crossover Linux. I get the same problems as with wine. To make things run, I borrowed a PC with XP to be able to operate the ?KTA, while I consider the WINE port now as hobby project and I consider to run install a virtual Windows on the Linux Machine. I could resolve an issue where Emain asked for some 32bit alsa- pulse.so files by installing a 32bit library, but now I am running into lots of page faults which have not been displayed before. Then the known messages about stack/heap problems and MFC42.DLL show up. Best reagrds, Wolfgang From moh_aldeeb from yahoo.com Sat Jan 3 23:07:53 2009 From: moh_aldeeb from yahoo.com (M Aldeeb) Date: Sun Jan 4 14:25:58 2009 Subject: PCR Primer Message-ID: <926275.79197.qm@web55803.mail.re3.yahoo.com> Dear All, ? I am looking for a good free online PCR primer design software. Thanks. ? Cheers!! ? Moh From charlesfracchia from gmail.com Sun Jan 4 21:28:02 2009 From: charlesfracchia from gmail.com (Charles Fracchia) Date: Sun Jan 4 23:15:24 2009 Subject: Clarification for LacZ gene in expression vectors References: Message-ID: <1f843b92-d291-4568-9966-850ce720fb4a@w24g2000prd.googlegroups.com> On Jan 2, 10:27?am, "vedha giri" wrote: > Dear Sir, > > If I Ligate my gene of interest in to the expression vector that do not have > the LacZ gene, what will be result of Blue /white selection test? > > How should I conformed my insert result? > > Regards > > -- > K.Vedhagiri > Research Scholar, > Dr K.Natarajseenivasan Lab, > Department of Microbiology, > Bharathidasan University, > Tiruchirappalli - 620 024. > India. > Mobile +91 99446-83728 The LacZ gene from the lactose operon (http://en.wikipedia.org/wiki/ Lac_operon) encodes from the enzyme Beta-galactosidase. The point of Blue/White screening is to test for the activity of Beta- Galactosidase. In the Blue/White screening what you do is feed the bacterium X-Gal and if the enzyme is active it will create -as a product of the reaction- an insoluble blue compound resulting in the formation of blue colonies. The results will vary depending on what you want to do. It looks like you want to test for the uptake of your expression vector by the bacterium. First you need to make sure that the bacterial strain you are going to use does not have a functional lacZ gene in it (you could do that with a White/Blue screening). At this point your bacteria should all be white. You can subculture one of the white colonies and incubate to use for your transfection. Once you have created your ligated the gene of interest to your expression vector containing a functional copy of the LacZ gene -which itself is linked to a promoter-, transfect your cells. Now you can perform a valid White/Blue test providing you are under conditions that promote the expression of lacZ (depends on the promoter you link it to) The colonies that have succesfully uptaken your expression vector should be blue. Hope this answers your questions. From dee from xray.bmc.uu.se Sun Jan 4 16:06:02 2009 From: dee from xray.bmc.uu.se (YU XIAODI) Date: Sun Jan 4 23:15:31 2009 Subject: PCR Primer In-Reply-To: <926275.79197.qm@web55803.mail.re3.yahoo.com> References: <926275.79197.qm@web55803.mail.re3.yahoo.com> Message-ID: Vecter NTI it is free for educations On Jan 4, 2009, at 5:07 AM, M Aldeeb wrote: > Dear All, > > I am looking for a good free online PCR primer design software. > Thanks. > > Cheers!! > > Moh > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From R.Jayakumar from roswellpark.org Sun Jan 4 17:18:48 2009 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Sun Jan 4 23:15:48 2009 Subject: PCR Primer References: <926275.79197.qm@web55803.mail.re3.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E23716D23@VISTA.roswellpark.org> Google for Primer3. It is really good. Jay //////////////////////////////\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Jayakumar R. Nair, Ph.D. Dept. of Immunology Roswell Park Cancer Institute Buffalo, NY 14263 Lab: 716-845-8231 Tel: 716-510-7961 Email: r.jayakumar@roswellpark.org ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of M Aldeeb Sent: Sat 1/3/2009 11:07 PM To: Methods Subject: PCR Primer Dear All, I am looking for a good free online PCR primer design software. Thanks. Cheers!! Moh _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods This email message may contain legally privileged and/or confidential information. If you are not the intended recipient(s), or the employee or agent responsible for the delivery of this message to the intended recipient(s), you are hereby notified that any disclosure, copying, distribution, or use of this email message is prohibited. If you have received this message in error, please notify the sender immediately by e-mail and delete this email message from your computer. Thank you. From lautys from gmail.com Sun Jan 4 19:45:53 2009 From: lautys from gmail.com (lautys) Date: Sun Jan 4 23:16:05 2009 Subject: PCR Primer In-Reply-To: <926275.79197.qm@web55803.mail.re3.yahoo.com> References: <926275.79197.qm@web55803.mail.re3.yahoo.com> Message-ID: Dear Moh, Primer 3: http://frodo.wi.mit.edu/. Good luck. Lau On Sun, Jan 4, 2009 at 12:07 PM, M Aldeeb wrote: > Dear All, > > I am looking for a good free online PCR primer design software. Thanks. > > Cheers!! > > Moh > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From quangtranho from yahoo.com Sun Jan 4 20:17:14 2009 From: quangtranho from yahoo.com (TRAN HO QUANG) Date: Sun Jan 4 23:16:41 2009 Subject: PCR Primer References: <926275.79197.qm@web55803.mail.re3.yahoo.com> Message-ID: <612872.76872.qm@web30405.mail.mud.yahoo.com> Hi Moh Try this http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Cheer Quang ----- Original Message ---- From: M Aldeeb To: Methods Sent: Sunday, January 4, 2009 11:07:53 AM Subject: PCR Primer Dear All, I am looking for a good free online PCR primer design software. Thanks. Cheers!! Moh _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From ddloeb from gmail.com Sun Jan 4 21:30:01 2009 From: ddloeb from gmail.com (ddloeb@gmail.com) Date: Sun Jan 4 23:17:09 2009 Subject: PCR Primer References: Message-ID: <3c513727-9fbc-4bb0-8d4e-b2b701ac0fce@g1g2000pra.googlegroups.com> On Jan 3, 10:07?pm, M Aldeeb wrote: > Dear All, > ? > I am looking for a good free online PCR primer design software. Thanks. > ? > Cheers!! > ? > Moh For what purpose? For regular PCR, we use Primer3 . For real-time QPCR, we use the RealTimeDesign program available at BioSearch Technologies . You need to setup a user account to use RealTimeDesign, but its free. Dan From prae from gmx.net Tue Jan 6 03:54:15 2009 From: prae from gmx.net (Christian Praetorius) Date: Tue Jan 6 13:05:11 2009 Subject: PCR Primer References: Message-ID: <6sgkhjF5tkjjU1@mid.individual.net> M Aldeeb wrote: >I am looking for a good free online PCR primer design software. Thanks. Primer3 has already been mentioned, another very good tool is PerlPrimer: http://perlprimer.sourceforge.net/ Christian -- X-no-Sig: yes From yvonne.couch from pharm.ox.ac.uk Tue Jan 6 12:03:39 2009 From: yvonne.couch from pharm.ox.ac.uk (stormgirlz) Date: Tue Jan 6 13:26:41 2009 Subject: Purity Message-ID: <21314438.post@talk.nabble.com> Hi all, relatively simple and silly question, if I get a compound at 90% purity, how do I compensate for that in my experiments? Cheers Storm -- View this message in context: http://www.nabble.com/Purity-tp21314438p21314438.html Sent from the Bio.net - Methods mailing list archive at Nabble.com. From pjie2 from cam.ac.uk Tue Jan 6 15:17:21 2009 From: pjie2 from cam.ac.uk (Peter Ellis) Date: Tue Jan 6 17:19:31 2009 Subject: Purity In-Reply-To: References: Message-ID: <6shsirF642fhU1@mid.individual.net> stormgirlz wrote: > Hi all, relatively simple and silly question, if I get a compound at 90% > purity, how do I compensate for that in my experiments? At the risk of stating the blindingly obvious, it depends what the other 10% is. If you know it's something completely inert, then you can just scale up the amount you put in accordingly. i.e. in order to add 1 gram of the stuff you want, you actually need to put in 1.111 grams of the impure stuff. If the other 10% has some sort of activity in your system, then you need to know what it is and work around it. If the impurities are sufficiently nasty, then the whole thing may be unusable. For example, if I ordered an oligonucleotide and it arrived as 90% DNA, 10% bleach, I wouldn't exactly run a PCR with it! If you have no idea what the other 10% is, then neither do we, and so we can't help you. Peter From I.Severin from nioo.knaw.nl Wed Jan 7 05:06:43 2009 From: I.Severin from nioo.knaw.nl (Severin, Ina) Date: Wed Jan 7 12:06:22 2009 Subject: plasmid standard curve qPCR Message-ID: <65F6E1EC64DCA6489800C09A2007FC6E02B7043B@cememail1.nioo.int> Hi all, I am currently working on the quantification of gene copy numbers and transcripts and got to the point where I need to make my standard curve. I have already isolated and linearized the plasmids containing the insert of interest but am (already for a while) wondering for what exact reason the linearization needs to be done. Unfortunately, I did not find much information in the literature (at least not in ecological research). Do the different formations of a plasmid behave differently in a qPCR reaction? And in how far could the buffer used for the digest interfere with my qPCR reaction? Do I need to clean my plasmid solution (and if yes, what's the best way)? Many thanks in advance! Ina From prae from gmx.net Wed Jan 7 03:50:40 2009 From: prae from gmx.net (Christian Praetorius) Date: Wed Jan 7 12:06:50 2009 Subject: Purity References: Message-ID: <6sj8n0F6a1njU1@mid.individual.net> stormgirlz wrote: >Hi all, relatively simple and silly question, if I get a compound at 90% >purity, how do I compensate for that in my experiments? It's a question, if this really matters. I would say, it depends on the type of compound and experiment, that you are doing. Christian -- X-no-Sig: yes From prae from gmx.net Wed Jan 7 03:53:19 2009 From: prae from gmx.net (Christian Praetorius) Date: Wed Jan 7 12:06:56 2009 Subject: Plates for ABI7500 Message-ID: <6sj8rvF6a1njU2@mid.individual.net> Hi, we are using our ABI7500 PCR cycler quite a lot. Since the plates, which are supplied by ABI are quite expensive does anyone know a good source for plates that can be used (this also applies for the foils used to seal the plates)? Or does it simply not matter and all I have to do is convince my colleagues about it? Christian -- X-no-Sig: yes From k.vd.wetering from nki.nl Wed Jan 7 04:51:54 2009 From: k.vd.wetering from nki.nl (| |Koen) Date: Wed Jan 7 12:07:02 2009 Subject: gateway cloning Message-ID: <1f02e40a-29f6-49dc-b4fe-61f512494511@a12g2000pro.googlegroups.com> Hi, we are currently using the Invitrogen Gateway system to make expression vectors for our genes of interest. We do so by using pDonr223 and our PCR amplified cDNA containing the recombination sites. For some genes this works well but for others we have difficulty in getting colonies containing the cDNA of interest. Our problem does not seem to be due to the length of the cDNA as we have succesfully made Entry clones containing cDNAs up to 5000 bp without any problem. Any ideas about the reseans for the varying efficiency of generating Entry vectors using the pDonr and BP-clonase 2 and the PCR amplified fragment containing recombination sites? Koen From aawara from pontiff-playground.org Wed Jan 7 05:05:33 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Wed Jan 7 12:07:07 2009 Subject: Plates for ABI7500 References: <6sj8rvF6a1njU2@mid.individual.net> Message-ID: In <6sj8rvF6a1njU2@mid.individual.net>, Christian Praetorius wrote: > we are using our ABI7500 PCR cycler quite a lot. Since the plates, > which are supplied by ABI are quite expensive does anyone know a good > source for plates that can be used (this also applies for the foils > used to seal the plates)? Or does it simply not matter and all I have > to do is convince my colleagues about it? Take a look at this PDF file to find plates that are compatible with your application: http://thelabshark.com/userfiles/File/Thermal_Cycler_Compatibility_Chart.pdf We use Pryme AVRT1 plates with the 7300 and 7500, but we are not doing FAST QPCR. From our vendor, the plates are $50 for 25 plates. There are likely to be cheaper vendors. For optical grade sealing film we pay $80 for 100 seals. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From chemdude321 from gmail.com Thu Jan 8 09:31:46 2009 From: chemdude321 from gmail.com (Josh Levin) Date: Thu Jan 8 12:23:55 2009 Subject: Primer design software Message-ID: In response to the posting regarding primer design software, here is a website of a company that I used to work for, Celadon Laboratories. They design a very user-friendly and comprehensive web-based primer design software: http://www.celadonlabs.com Josh From blackhole from abuse.plus.com Thu Jan 8 10:13:54 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Jan 8 12:24:06 2009 Subject: plasmid standard curve qPCR References: Message-ID: <9TSyNqSyghZJFAwt@abuse.plus.com> Historians believe that in newspost on Wed, 7 Jan 2009, "Severin, Ina" penned the following literary masterpiece: >I have already isolated and linearized the plasmids containing the >insert of interest but am (already for a while) wondering for what exact >reason the linearization needs to be done. Unfortunately, I did not find >much information in the literature (at least not in ecological >research). Do the different formations of a plasmid behave differently >in a qPCR reaction? Supercoiled plasmid is not ideal as it can be refractory to denaturation. Therefore one plays safe with linearisation. >And in how far could the buffer used for the digest >interfere with my qPCR reaction? 1ug of plasmid of 4kb will be 2.32 x 10E11 copies. A 10,000 fold diln of that will have diluted out any possible buffer interference. >Do I need to clean my plasmid solution >(and if yes, what's the best way)? Anyway you want but it's probably not worth it. For more detail you can ask on the qpcrlistserv on Yahoogroups. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From stewjw from gmail.com Fri Jan 9 04:04:53 2009 From: stewjw from gmail.com (StewJW) Date: Fri Jan 9 12:36:25 2009 Subject: Plates for ABI7500 References: <6sj8rvF6a1njU2@mid.individual.net> Message-ID: <6b655de0-af47-4b44-978d-3a83d93cbb15@s1g2000prg.googlegroups.com> There are a few suppliers here at Fisher UK (part of ThermoFisher) we suggest Abgene plates see the qPCR plate compatability guide: http://www.abgene.com/Static_Pages.asp?page=26 Best, Stewart From stewjw from gmail.com Fri Jan 9 04:38:07 2009 From: stewjw from gmail.com (StewJW) Date: Fri Jan 9 12:36:38 2009 Subject: gateway cloning References: <1f02e40a-29f6-49dc-b4fe-61f512494511@a12g2000pro.googlegroups.com> Message-ID: Hi Koen, I don't claim to be an expert in this area, but have you tried linearising your attB expression clones as described in the Invitrogen manual. Also, have you purified the PCR product to remove attB primers and any attB primer-dimers. Invitrogen also recommends not to use phenol/chloroform extraction clean up methods. Like you say its unlikely to be a size issue which is more of a problem with making entry vectors using TA cloning methods where the upper limit is around 5kb. There is also something else nagging me about problems witrh certain sequences, which is unpublished but I can't remember what it is may be someone else can help here. Best, Stewart From stewjw from gmail.com Fri Jan 9 04:41:38 2009 From: stewjw from gmail.com (StewJW) Date: Fri Jan 9 12:36:47 2009 Subject: gateway cloning References: <1f02e40a-29f6-49dc-b4fe-61f512494511@a12g2000pro.googlegroups.com> Message-ID: <0f6e4f8b-5010-49f3-bf18-039531f1ac06@o4g2000pra.googlegroups.com> I should also add have you performed control reactions to check your reagents, occasionally there are problems with the clonase mixture, and Invitrogen provides controls in their kits. From fboernke from biologie.uni-erlangen.de Fri Jan 9 08:43:03 2009 From: fboernke from biologie.uni-erlangen.de (fboernke@biologie.uni-erlangen.de) Date: Fri Jan 9 12:37:00 2009 Subject: pHIS2.1 one-hybrid vector request Message-ID: Dear all, I'd like to do a yeast one-hybrid screen to indentify proteins binding to a specific DNA fragment. I've got all the neccesary components except the pHIS2.1 reporter vector which is part of clontech's matchmaker yeast one hybrid kit. Unfortunately, they do not sell this plasmid separately. Does anybody have this vector and would be willing to share an aliquot? Thanks in advance Ricky From molecular from khayam.ut.ac.ir Sun Jan 11 08:04:59 2009 From: molecular from khayam.ut.ac.ir (Mohadese DastPeyman) Date: Sun Jan 11 13:00:24 2009 Subject: adhesion assay Message-ID: Dear Dr.Zbigniew Rudzki I'm Mohadese Dastpeyman,MSC student of molecular cell biology at tehran university,I saw your direction to Sebastien PARIS about adhesion assay at this site http://www.bio.net/bionet/mm/methods/1998-August/069662.html as you are well experienced in adhesion assay would you please help me? My thesis is about effect of a herbal drug on metastasis and invasion properties of MDA-MB-231 breast cancer cells. I want to test cell adhesion assay for this cells with collagen type 2. now ,as you regarded before,there are a lot of protocols and I 'm wondering between two of them about counting of the remaining cells.some prptpcpls suggested adding MTT,and some of them suggested fixing cells with christal violet and then lysed cells with triton x 100 ,then read the absorbance at 550nm. your suggestion is important to me,what do you thimk ?which of them is better ? is there any protocol that you accepted it as a standardize protocol? With regards Mohadese Dastpeyman Department of molecular cell biology Tehran University of Sciences, Tehran, Iran, Tel:+989112454702 From r.srivastava from lrz.tu-muenchen.de Mon Jan 12 05:01:22 2009 From: r.srivastava from lrz.tu-muenchen.de (rashmi srivastava) Date: Mon Jan 12 14:13:37 2009 Subject: protocol for ATP-regenerating system Message-ID: <000301c9749c$b9383ac0$4865a8c0@Biopolymere.wzw.tum.de> Hi could someone please send me the protocol for ATP-regenerating system? Thanks, Rashmi From LKovatch from kbpllc.com Mon Jan 12 14:40:59 2009 From: LKovatch from kbpllc.com (Lori Kovatch) Date: Mon Jan 12 15:33:43 2009 Subject: pI determination methods for QC Message-ID: <923F206C7FF8914E852DBACDB61A06340578A027@EXCH02.ngenx.com> Hi, I am currently attempting to bring IEF testing in house for our Quality Control lab. We have two proteins that we need to be able to determine pI. I know around what the pI should be as we have sent them to a contract lab for testing previously. One of the proteins has a rather high pI around 11. I thought I could use Invitrogen's IPG strips with the Zoom Runner system, but I am unsure of how to actually determine the pI once I have run the system. Now from what I understand, their system is more a tool for a separation of proteins and not for the determination of pI. Does anyone know how I could determine or if I can determine pI using this system? Thanks Lori Kovatch Long Quality Control Manager Kentucky BioProcessing, LLC 3700 Airpark Drive Owensboro, KY 42301 ph 270-663-6132 fax 270-689-2571 From nobody from nospam.not Mon Jan 12 21:13:50 2009 From: nobody from nospam.not (Han) Date: Mon Jan 12 21:54:23 2009 Subject: DNA software question Message-ID: Colleague at Cornell Medical College has a very old Mac with an old version of DNAStar, which he likes very much. His use is not very intensive, but primer design, sequence alignment etc are used relatively often. Since the Mac is about to die, he would like software for a PC (XP Pro) that could best perform those functions. DNAStar (new version) costs $1,000/year in licensing costs, which colleague doesn't regard as a wise investment. What are his best options for free or cheap software with similar capabilities as what he wants of DNAStar (primer design, sequence alignment, etc)? -- Best regards Han email address is invalid From nick.theodorakis from gmail.com Mon Jan 12 22:04:12 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Tue Jan 13 13:22:02 2009 Subject: DNA software question References: Message-ID: Han wrote: > Colleague at Cornell Medical College has a very old Mac with an old version > of DNAStar, which he likes very much. His use is not very intensive, but > primer design, sequence alignment etc are used relatively often. > > Since the Mac is about to die, he would like software for a PC (XP Pro) > that could best perform those functions. DNAStar (new version) costs > $1,000/year in licensing costs, which colleague doesn't regard as a wise > investment. What are his best options for free or cheap software with > similar capabilities as what he wants of DNAStar (primer design, sequence > alignment, etc)? > For primer design, I and nearly everyone I know use Pimer3. There is a web version: which is also now built into the NCBI primer blast page: Downloadable source is also available: -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From Zhonglin.Chai from bakeridi.edu.au Mon Jan 12 22:16:42 2009 From: Zhonglin.Chai from bakeridi.edu.au (Zhonglin Chai) Date: Tue Jan 13 13:22:11 2009 Subject: DNA software question In-Reply-To: References: Message-ID: <217613C88C00D4448B099AD7A374F25302C971D0@exch01.bhri.internal> Try online softwares. You can start from this link: http://www.expasy.ch/tools/ Dr Zhonglin Chai, PhD -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Han Sent: Tuesday, 13 January 2009 01:14 PM To: methods@magpie.bio.indiana.edu Subject: DNA software question Colleague at Cornell Medical College has a very old Mac with an old version of DNAStar, which he likes very much. His use is not very intensive, but primer design, sequence alignment etc are used relatively often. Since the Mac is about to die, he would like software for a PC (XP Pro) that could best perform those functions. DNAStar (new version) costs $1,000/year in licensing costs, which colleague doesn't regard as a wise investment. What are his best options for free or cheap software with similar capabilities as what he wants of DNAStar (primer design, sequence alignment, etc)? -- Best regards Han email address is invalid _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________________ This email has been scanned by the MessageLabs Email Security System. ______________________________________________________________________ This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you have received this email in error please promptly delete this email and notify the system manager. This email has been scanned by the MessageLabs Email Security System. For more information please visit http://www.messagelabs.com/email From ivanoov from gmail.com Tue Jan 13 03:33:09 2009 From: ivanoov from gmail.com (chovek69) Date: Tue Jan 13 13:22:17 2009 Subject: DNA software question References: Message-ID: <43566c93-a960-4f46-97d5-5a265496504a@q35g2000vbi.googlegroups.com> On Jan 13, 5:04?am, Nick Theodorakis wrote: > Han wrote: > > Colleague at Cornell Medical College has a very old Mac with an old version > > of DNAStar, which he likes very much. ?His use is not very intensive, but > > primer design, sequence alignment etc are used relatively often. > > > Since the Mac is about to die, he would like software for a PC (XP Pro) > > that could best perform those functions. ?DNAStar (new version) costs > > $1,000/year in licensing costs, which colleague doesn't regard as a wise > > investment. ?What are his best options for free or cheap software with > > similar capabilities as what he wants of DNAStar (primer design, sequence > > alignment, etc)? > > For primer design, I and nearly everyone I know use Pimer3. There is a > web version: > > > > which is also now built into the NCBI primer blast page: > > LINK_LOC=BlastHome> > > Downloadable source is also available: > > > > -- > Nick Theodorakis > nick_theodora...@hotmail.com > contact form:http://theodorakis.net/contact.html and for seq alignment Bioedit and MEGA 4... From aawara from pontiff-playground.org Tue Jan 13 09:33:13 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Tue Jan 13 13:23:42 2009 Subject: DNA software question References: Message-ID: In , Han wrote: > Since the Mac is about to die, he would like software for a PC (XP Pro) > that could best perform those functions. DNAStar (new version) costs > $1,000/year in licensing costs, which colleague doesn't regard as a wise > investment. What are his best options for free or cheap software with > similar capabilities as what he wants of DNAStar (primer design, sequence > alignment, etc)? > We use "ApE" (A Plasmid Editor) and "Serial Cloner". ApE is available from: Serial Cloner is available from: Both are free, although a donation is suggested for Serial Cloner. ApE reads ABI sequencing trace files, does restriction digests, sequence alignments, finds primers, finds silent restriction endonuclease recognition sites, does translations, produces excellent annotated plasmid maps, and can BLAST sequences. Serial Cloner does everything that ApE does, and in addition, shRNA design, Gateway(tm) cloning design, and one can setup ligations of 1, 2, or 3 fragments. The plasmid maps generated by Serial Cloner are similar to those made by DNA Strider (i.e. restriction sites but not annotated). AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From eenigoy from gmail.com Wed Jan 14 03:44:39 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Wed Jan 14 14:19:56 2009 Subject: 2D gel analysis software Message-ID: <77ddf6c70901140044p545aed82q52a18dcb1e87dcbf@mail.gmail.com> Hello! What exactly should one look for while selecting a particular 2D gel analysis software? Can people share their experiences about the soft wares they are using? Regards, -- Yg -- Yoginee Budhkar Junior Research Fellow Food Engineering and Technology Department Institute of Chemical Technology Matunga, Mumbai 400019 Contact: yogineeb.foodbio@udct.org From yasirphr from yahoo.com Tue Jan 13 20:04:38 2009 From: yasirphr from yahoo.com (muhammad yasir) Date: Wed Jan 14 14:25:00 2009 Subject: Low concentration miniprep In-Reply-To: <200901131706.n0DH64813679@net.bio.net> Message-ID: <161483.83541.qm@web55906.mail.re3.yahoo.com> I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep. ? Yasir From ivanoov from gmail.com Thu Jan 15 03:08:59 2009 From: ivanoov from gmail.com (chovek69) Date: Thu Jan 15 10:21:45 2009 Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR Message-ID: Dear experts, I am relatively new in the Real-time PCR world. I am about to order a bunch of hydrolysis probes quenched with the BHQ. The company states exclusively that they can do 3' - BHQ labeling but is this also mean that the 3'-OH will be blocked by the quencher against extension or I must order 3' phosphorylation in addition. Any suggestions will be most welcome... Thanks Ivan From fulvio.celsi from gmail.com Thu Jan 15 06:46:31 2009 From: fulvio.celsi from gmail.com (Fulvio Celsi) Date: Thu Jan 15 10:22:13 2009 Subject: Low concentration miniprep In-Reply-To: <161483.83541.qm@web55906.mail.re3.yahoo.com> References: <200901131706.n0DH64813679@net.bio.net> <161483.83541.qm@web55906.mail.re3.yahoo.com> Message-ID: Hi Yasir is your gene very large?what kit are you using? if the gene (and so the plasmid) is large, maybe you should try to increase the quantity of the bacteria when doing miniprep... 2009/1/14 muhammad yasir > I have clone the gene in pUC19 and get conformation with restriction > digestion. But unfortunately, I get very low quantity of plasmid after > miniprep. While the control (circular pUC19) concentration is quite enough > with miniprep kit that I used. I shall be very glad to know about the > possible reason of low concentration and how can I improve the yield of > clone plasmid from miniprep. > > Yasir > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Fulvio Celsi BsC,PhD Sector of Neurobiology, International School for Advanced Studies, Scuola Internazionale di Studi Superiori Avanzati (SISSA), Trieste Italy Mobile: +393286489131 From davidminde from gmail.com Thu Jan 15 10:41:28 2009 From: davidminde from gmail.com (David-Paul Minde) Date: Thu Jan 15 11:54:58 2009 Subject: Low concentration miniprep In-Reply-To: References: <200901131706.n0DH64813679@net.bio.net> <161483.83541.qm@web55906.mail.re3.yahoo.com> Message-ID: <123243900901150741jaa1901p96f0c9f261b153fe@mail.gmail.com> Hi Yasir > is your gene very large?what kit are you using? if the gene (and so the > plasmid) is large, maybe you should try to increase the quantity of the > bacteria when doing miniprep... e.g. Terrific Broth would help in this case (and if still necessary) bigger volumes of culture. Of course you shold not exceed the specs of your kit (or you will have decrease of DNA quality) best, David > > > 2009/1/14 muhammad yasir > > > I have clone the gene in pUC19 and get conformation with restriction > > digestion. But unfortunately, I get very low quantity of plasmid after > > miniprep. While the control (circular pUC19) concentration is quite > enough > > with miniprep kit that I used. I shall be very glad to know about the > > possible reason of low concentration and how can I improve the yield of > > clone plasmid from miniprep. > > > > Yasir > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Fulvio Celsi > BsC,PhD > Sector of Neurobiology, > International School for Advanced Studies, Scuola Internazionale di Studi > Superiori Avanzati (SISSA), > Trieste > Italy > > Mobile: +393286489131 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht I never think of the future. It comes soon enough. Albert Einstein Res severa verum gaudium. (~true delight is a severe issue) Seneca From hutsugi from fhcrc.org Thu Jan 15 13:16:55 2009 From: hutsugi from fhcrc.org (Utsugi, Heidi K) Date: Thu Jan 15 17:00:46 2009 Subject: Low concentration miniprep References: <200901151704.n0FH46817909@net.bio.net> Message-ID: "I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep. Yasir" >From what I have seen, more often that not, overloading a commercial DNA miniprep column is very likely. This may be copy number related. The next culprit is overgrown plates with satellite colonies picked instead of transformed. -- Heidi ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu Sent: Thu 1/15/2009 9:04 AM To: methods@magpie.bio.indiana.edu Subject: Methods Digest, Vol 44, Issue 13 Send Methods mailing list submissions to methods@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/methods or, via email, send a message with subject or body 'help' to methods-request@net.bio.net You can reach the person managing the list at methods-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Methods digest..." Today's Topics: 1. 2D gel analysis software (yoginee budhkar) 2. Low concentration miniprep (muhammad yasir) 3. Re: Low concentration miniprep (Fulvio Celsi) 4. Black hole quenchers (BHQ) and 3' blocking in Real-time PCR (chovek69) 5. Re: Low concentration miniprep (David-Paul Minde) ---------------------------------------------------------------------- Message: 1 Date: Wed, 14 Jan 2009 14:14:39 +0530 From: "yoginee budhkar" Subject: 2D gel analysis software To: methods@magpie.bio.indiana.edu Message-ID: <77ddf6c70901140044p545aed82q52a18dcb1e87dcbf@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 Hello! What exactly should one look for while selecting a particular 2D gel analysis software? Can people share their experiences about the soft wares they are using? Regards, -- Yg -- Yoginee Budhkar Junior Research Fellow Food Engineering and Technology Department Institute of Chemical Technology Matunga, Mumbai 400019 Contact: yogineeb.foodbio@udct.org ------------------------------ Message: 2 Date: Tue, 13 Jan 2009 17:04:38 -0800 (PST) From: muhammad yasir Subject: Low concentration miniprep To: methods@oat.bio.indiana.edu Message-ID: <161483.83541.qm@web55906.mail.re3.yahoo.com> Content-Type: text/plain; charset=iso-8859-1 I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep. Yasir ------------------------------ Message: 3 Date: Thu, 15 Jan 2009 12:46:31 +0100 From: "Fulvio Celsi" Subject: Re: Low concentration miniprep To: yasirphr@yahoo.com Cc: methods@oat.bio.indiana.edu Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Hi Yasir is your gene very large?what kit are you using? if the gene (and so the plasmid) is large, maybe you should try to increase the quantity of the bacteria when doing miniprep... 2009/1/14 muhammad yasir > I have clone the gene in pUC19 and get conformation with restriction > digestion. But unfortunately, I get very low quantity of plasmid after > miniprep. While the control (circular pUC19) concentration is quite enough > with miniprep kit that I used. I shall be very glad to know about the > possible reason of low concentration and how can I improve the yield of > clone plasmid from miniprep. > > Yasir > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Fulvio Celsi BsC,PhD Sector of Neurobiology, International School for Advanced Studies, Scuola Internazionale di Studi Superiori Avanzati (SISSA), Trieste Italy Mobile: +393286489131 ------------------------------ Message: 4 Date: Thu, 15 Jan 2009 00:08:59 -0800 (PST) From: chovek69 Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR To: methods@net.bio.net Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Dear experts, I am relatively new in the Real-time PCR world. I am about to order a bunch of hydrolysis probes quenched with the BHQ. The company states exclusively that they can do 3' - BHQ labeling but is this also mean that the 3'-OH will be blocked by the quencher against extension or I must order 3' phosphorylation in addition. Any suggestions will be most welcome... Thanks Ivan ------------------------------ Message: 5 Date: Thu, 15 Jan 2009 16:41:28 +0100 From: "David-Paul Minde" Subject: Re: Low concentration miniprep To: "Fulvio Celsi" Cc: methods@oat.bio.indiana.edu, yasirphr@yahoo.com Message-ID: <123243900901150741jaa1901p96f0c9f261b153fe@mail.gmail.com> Content-Type: text/plain; charset=UTF-8 Hi Yasir > is your gene very large?what kit are you using? if the gene (and so the > plasmid) is large, maybe you should try to increase the quantity of the > bacteria when doing miniprep... e.g. Terrific Broth would help in this case (and if still necessary) bigger volumes of culture. Of course you shold not exceed the specs of your kit (or you will have decrease of DNA quality) best, David > > > 2009/1/14 muhammad yasir > > > I have clone the gene in pUC19 and get conformation with restriction > > digestion. But unfortunately, I get very low quantity of plasmid after > > miniprep. While the control (circular pUC19) concentration is quite > enough > > with miniprep kit that I used. I shall be very glad to know about the > > possible reason of low concentration and how can I improve the yield of > > clone plasmid from miniprep. > > > > Yasir > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Fulvio Celsi > BsC,PhD > Sector of Neurobiology, > International School for Advanced Studies, Scuola Internazionale di Studi > Superiori Avanzati (SISSA), > Trieste > Italy > > Mobile: +393286489131 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- David Minde MSc (TUM) Cellular Protein Chemistry Room 707 Department of Chemistry Faculty of Science Utrecht University Krytgebouw Padualaan 8 NL-3584 CH Utrecht The Netherlands office phone +31 30 253 4105 mobile phone +31(0)631154267 private address: Griftkade 4 bis 3572 TW Utrecht I never think of the future. It comes soon enough. Albert Einstein Res severa verum gaudium. (~true delight is a severe issue) Seneca ------------------------------ _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods End of Methods Digest, Vol 44, Issue 13 *************************************** From Sharon.Waldrop from utsouthwestern.edu Thu Jan 15 15:06:10 2009 From: Sharon.Waldrop from utsouthwestern.edu (Sharon Waldrop) Date: Thu Jan 15 17:01:06 2009 Subject: Eukaryotic Cell Membrane Specific Vector Message-ID: <496F42D30200003E00042153@swnw126.swmed.org> Hi Everyone, I need a vector that is membrane specific for my control cell line. When I use the HEK293 cell line (control - does not have receptor in membrane or else where), after transfection I have a diffuse cytoplasmic EGFP+. When I use the HTC cell line (experimental - receptor is present in the membrane), I have a specific membrane EGFP+ product. I am currently using pIRES2-EGFP. Any help is appreciated. Thanks, Shar From blackhole from abuse.plus.com Fri Jan 16 10:05:24 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Fri Jan 16 12:07:52 2009 Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR References: Message-ID: Historians believe that in newspost on Thu, 15 Jan 2009, chovek69 penned the following literary masterpiece: >The company states exclusively that they can do 3' - BHQ labeling but >is this also mean that the 3'-OH will be blocked by the quencher >against extension or I must order 3' phosphorylation in addition. No need for 3' phosphorylation. They will use a column that has quencher attached. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From nobody from nospam.not Fri Jan 16 21:19:44 2009 From: nobody from nospam.not (Han) Date: Fri Jan 16 23:15:01 2009 Subject: DNA software question References: Message-ID: Thanks all for your advice. It's greatly appreciated. -- Best regards Han email address is invalid From yasirphr from yahoo.com Fri Jan 16 19:56:14 2009 From: yasirphr from yahoo.com (muhammad yasir) Date: Fri Jan 16 23:23:33 2009 Subject: Low concentration miniprep In-Reply-To: <200901161705.n0GH55823292@net.bio.net> Message-ID: <169375.19575.qm@web55908.mail.re3.yahoo.com> - From shifalich from rediffmail.com Sat Jan 17 01:45:43 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Sat Jan 17 16:25:38 2009 Subject: Low concentration miniprep Message-ID: <20090117064543.47791.qmail@f4mail-234-123.rediffmail.com> ?Just a query! Are satellite colonies correctly defined as slow growing colonies or a daugther colony of nearby parent colony? Additionally, I once picked up satellite colonies, they never grew in LB medium. HOwever, Yasir can try increasing the volume of medium for inoculation and getting a bigger pellet,keeping all other solutions' volume to be the same. Shifali On Thu, 15 Jan 2009 Utsugi,Heidi K wrote : >"I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep. > >Yasir" > > >From what I have seen, more often that not, overloading a commercial DNA miniprep column is very likely. This may be copy number related. The next culprit is overgrown plates with satellite colonies picked instead of transformed. -- Heidi > > >________________________________ > > From: methods-bounces@oat.bio.indiana.edu on behalf of methods-request@oat.bio.indiana.edu >Sent: Thu 1/15/2009 9:04 AM >To: methods@magpie.bio.indiana.edu >Subject: Methods Digest, Vol 44, Issue 13 > > > >Send Methods mailing list submissions to > methods@net.bio.net > >To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods >or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > >You can reach the person managing the list at > methods-owner@net.bio.net > >When replying, please edit your Subject line so it is more specific >than "Re: Contents of Methods digest..." > > >Today's Topics: > > 1. 2D gel analysis software (yoginee budhkar) > 2. Low concentration miniprep (muhammad yasir) > 3. Re: Low concentration miniprep (Fulvio Celsi) > 4. Black hole quenchers (BHQ) and 3' blocking in Real-time PCR > (chovek69) > 5. Re: Low concentration miniprep (David-Paul Minde) > > >---------------------------------------------------------------------- > >Message: 1 >Date: Wed, 14 Jan 2009 14:14:39 +0530 > From: "yoginee budhkar" >Subject: 2D gel analysis software >To: methods@magpie.bio.indiana.edu >Message-ID: > <77ddf6c70901140044p545aed82q52a18dcb1e87dcbf@mail.gmail.com> >Content-Type: text/plain; charset=ISO-8859-1 > >Hello! > >What exactly should one look for while selecting a particular 2D gel >analysis software? Can people share their experiences about the soft wares >they are using? > >Regards, >-- Yg >-- >Yoginee Budhkar >Junior Research Fellow >Food Engineering and Technology Department >Institute of Chemical Technology >Matunga, Mumbai 400019 >Contact: yogineeb.foodbio@udct.org > > >------------------------------ > >Message: 2 >Date: Tue, 13 Jan 2009 17:04:38 -0800 (PST) > From: muhammad yasir >Subject: Low concentration miniprep >To: methods@oat.bio.indiana.edu >Message-ID: <161483.83541.qm@web55906.mail.re3.yahoo.com> >Content-Type: text/plain; charset=iso-8859-1 > >I have clone the gene in pUC19 and get conformation with restriction digestion. But unfortunately, I get very low quantity of plasmid after miniprep. While the control (circular pUC19) concentration is quite enough with miniprep kit that I used. I shall be very glad to know about the possible reason of low concentration and how can I improve the yield of clone plasmid from miniprep. > >Yasir > > > > >------------------------------ > >Message: 3 >Date: Thu, 15 Jan 2009 12:46:31 +0100 > From: "Fulvio Celsi" >Subject: Re: Low concentration miniprep >To: yasirphr@yahoo.com >Cc: methods@oat.bio.indiana.edu >Message-ID: > >Content-Type: text/plain; charset=ISO-8859-1 > >Hi Yasir >is your gene very large?what kit are you using? if the gene (and so the >plasmid) is large, maybe you should try to increase the quantity of the >bacteria when doing miniprep... > >2009/1/14 muhammad yasir > > > I have clone the gene in pUC19 and get conformation with restriction > > digestion. But unfortunately, I get very low quantity of plasmid after > > miniprep. While the control (circular pUC19) concentration is quite enough > > with miniprep kit that I used. I shall be very glad to know about the > > possible reason of low concentration and how can I improve the yield of > > clone plasmid from miniprep. > > > > Yasir > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > >-- >Fulvio Celsi >BsC,PhD >Sector of Neurobiology, >International School for Advanced Studies, Scuola Internazionale di Studi >Superiori Avanzati (SISSA), >Trieste >Italy > >Mobile: +393286489131 > > >------------------------------ > >Message: 4 >Date: Thu, 15 Jan 2009 00:08:59 -0800 (PST) > From: chovek69 >Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR >To: methods@net.bio.net >Message-ID: > >Content-Type: text/plain; charset=ISO-8859-1 > >Dear experts, > >I am relatively new in the Real-time PCR world. I am about to order a >bunch of hydrolysis probes quenched with the BHQ. > >The company states exclusively that they can do 3' - BHQ labeling but >is this also mean that the 3'-OH will be blocked by the quencher >against extension or I must order 3' phosphorylation in addition. > >Any suggestions will be most welcome... >Thanks >Ivan > > >------------------------------ > >Message: 5 >Date: Thu, 15 Jan 2009 16:41:28 +0100 > From: "David-Paul Minde" >Subject: Re: Low concentration miniprep >To: "Fulvio Celsi" >Cc: methods@oat.bio.indiana.edu, yasirphr@yahoo.com >Message-ID: > <123243900901150741jaa1901p96f0c9f261b153fe@mail.gmail.com> >Content-Type: text/plain; charset=UTF-8 > >Hi Yasir > > > is your gene very large?what kit are you using? if the gene (and so the > > plasmid) is large, maybe you should try to increase the quantity of the > > bacteria when doing miniprep... > >e.g. Terrific Broth would help in this case (and if still necessary) bigger >volumes of culture. >Of course you shold not exceed the specs of your kit (or you will have >decrease of DNA quality) >best, >David > > > > > > > 2009/1/14 muhammad yasir > > > > > I have clone the gene in pUC19 and get conformation with restriction > > > digestion. But unfortunately, I get very low quantity of plasmid after > > > miniprep. While the control (circular pUC19) concentration is quite > > enough > > > with miniprep kit that I used. I shall be very glad to know about the > > > possible reason of low concentration and how can I improve the yield of > > > clone plasmid from miniprep. > > > > > > Yasir > > > > > > > > > > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > > > -- > > Fulvio Celsi > > BsC,PhD > > Sector of Neurobiology, > > International School for Advanced Studies, Scuola Internazionale di Studi > > Superiori Avanzati (SISSA), > > Trieste > > Italy > > > > Mobile: +393286489131 > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > >-- >David Minde MSc (TUM) > >Cellular Protein Chemistry Room 707 >Department of Chemistry >Faculty of Science >Utrecht University >Krytgebouw >Padualaan 8 >NL-3584 CH Utrecht >The Netherlands > >office phone +31 30 253 4105 >mobile phone +31(0)631154267 > >private address: >Griftkade 4 bis >3572 TW Utrecht > >I never think of the future. It comes soon enough. > Albert Einstein >Res severa verum gaudium. (~true delight is a severe issue) > Seneca > > >------------------------------ > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > >End of Methods Digest, Vol 44, Issue 13 >*************************************** > > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From ivanoov from gmail.com Sat Jan 17 09:08:13 2009 From: ivanoov from gmail.com (chovek69) Date: Sat Jan 17 17:15:47 2009 Subject: Black hole quenchers (BHQ) and 3' blocking in Real-time PCR References: Message-ID: <5b159437-465c-4b03-9d5d-3588f459261f@p2g2000prf.googlegroups.com> On Jan 16, 5:05?pm, Duncan Clark wrote: > Historians believe that in newspost > on > Thu, 15 Jan 2009, chovek69 penned the following > literary masterpiece: > > >The company states exclusively that they can do 3' - BHQ labeling but > >is this also mean that the 3'-OH will be blocked by the quencher > >against extension ?or I must order 3' phosphorylation in addition. > > No need for 3' phosphorylation. They will use a column that has quencher > attached. > > Duncan > -- > I love deadlines. I especially like the whooshing noise they make as > they go flying by. > > Duncan Clark > GeneSys Ltd. Thanks alot Duncan .... your suggestions are always the best From virashkgupta from gmail.com Sat Jan 17 00:19:58 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Jan 17 17:15:57 2009 Subject: Methods Digest, Vol 44, Issue 12 In-Reply-To: <200901141704.n0EH4d801927@net.bio.net> References: <200901141704.n0EH4d801927@net.bio.net> Message-ID: FastPCR from primer digital ltd is freeware and complete solution for primer designing with excellent additional features. To academic institutions FastPCR is available for a free, provided for non-commercial research and education use only (not for reproduction or distribution, cannot be used in any commercial enterprises). The manual, licence and files for installation are available over the Internet at: http://www.biocenter.helsinki.fi/bi/programs/fastpcr.htm or from PrimerDigital Ltd: http://www.primerdigital.com/ all the best On 1/14/09, methods-request@oat.bio.indiana.edu < methods-request@oat.bio.indiana.edu> wrote: > > Send Methods mailing list submissions to > methods@net.bio.net > > To subscribe or unsubscribe via the World Wide Web, visit > http://www.bio.net/biomail/listinfo/methods > or, via email, send a message with subject or body 'help' to > methods-request@net.bio.net > > You can reach the person managing the list at > methods-owner@net.bio.net > > When replying, please edit your Subject line so it is more specific > than "Re: Contents of Methods digest..." > > > Today's Topics: > > 1. Re: DNA software question (Nick Theodorakis) > 2. RE: DNA software question (Zhonglin Chai) > 3. Re: DNA software question (chovek69) > 4. Re: DNA software question (Aawara Chowdhury) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 12 Jan 2009 19:04:12 -0800 (PST) > From: Nick Theodorakis > Subject: Re: DNA software question > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > > > Han wrote: > > Colleague at Cornell Medical College has a very old Mac with an old > version > > of DNAStar, which he likes very much. His use is not very intensive, but > > primer design, sequence alignment etc are used relatively often. > > > > Since the Mac is about to die, he would like software for a PC (XP Pro) > > that could best perform those functions. DNAStar (new version) costs > > $1,000/year in licensing costs, which colleague doesn't regard as a wise > > investment. What are his best options for free or cheap software with > > similar capabilities as what he wants of DNAStar (primer design, sequence > > alignment, etc)? > > > > For primer design, I and nearly everyone I know use Pimer3. There is a > web version: > > > > which is also now built into the NCBI primer blast page: > > LINK_LOC=BlastHome> > > Downloadable source is also available: > > > > > -- > Nick Theodorakis > nick_theodorakis@hotmail.com > contact form: > http://theodorakis.net/contact.html > > > ------------------------------ > > Message: 2 > Date: Tue, 13 Jan 2009 14:16:42 +1100 > From: "Zhonglin Chai" > Subject: RE: DNA software question > To: "Han" , > Message-ID: > <217613C88C00D4448B099AD7A374F25302C971D0@exch01.bhri.internal> > Content-Type: text/plain; charset="us-ascii" > > Try online softwares. You can start from this link: > http://www.expasy.ch/tools/ > > Dr Zhonglin Chai, PhD > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu > [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Han > Sent: Tuesday, 13 January 2009 01:14 PM > To: methods@magpie.bio.indiana.edu > Subject: DNA software question > > Colleague at Cornell Medical College has a very old Mac with an old > version of DNAStar, which he likes very much. His use is not very > intensive, but primer design, sequence alignment etc are used relatively > often. > > Since the Mac is about to die, he would like software for a PC (XP Pro) > that could best perform those functions. DNAStar (new version) costs > $1,000/year in licensing costs, which colleague doesn't regard as a wise > investment. What are his best options for free or cheap software with > similar capabilities as what he wants of DNAStar (primer design, > sequence alignment, etc)? > > -- > Best regards > Han > email address is invalid > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > ______________________________________________________________________ > This email has been scanned by the MessageLabs Email Security System. > ______________________________________________________________________ > > This email and any files transmitted with it are confidential and intended > solely for the use of the individual or entity to whom they are addressed. > If you have received this email in error please promptly delete this email > and notify the system manager. > > This email has been scanned by the MessageLabs Email Security System. > For more information please visit http://www.messagelabs.com/email > > > > ------------------------------ > > Message: 3 > Date: Tue, 13 Jan 2009 00:33:09 -0800 (PST) > From: chovek69 > Subject: Re: DNA software question > To: methods@net.bio.net > Message-ID: > <43566c93-a960-4f46-97d5-5a265496504a@q35g2000vbi.googlegroups.com> > Content-Type: text/plain; charset=ISO-8859-1 > > On Jan 13, 5:04 am, Nick Theodorakis > wrote: > > Han wrote: > > > Colleague at Cornell Medical College has a very old Mac with an old > version > > > of DNAStar, which he likes very much. His use is not very intensive, > but > > > primer design, sequence alignment etc are used relatively often. > > > > > Since the Mac is about to die, he would like software for a PC (XP Pro) > > > that could best perform those functions. DNAStar (new version) costs > > > $1,000/year in licensing costs, which colleague doesn't regard as a > wise > > > investment. What are his best options for free or cheap software with > > > similar capabilities as what he wants of DNAStar (primer design, > sequence > > > alignment, etc)? > > > > For primer design, I and nearly everyone I know use Pimer3. There is a > > web version: > > > > > > > > which is also now built into the NCBI primer blast page: > > > > > LINK_LOC=BlastHome> > > > > Downloadable source is also available: > > > > > > > > -- > > Nick Theodorakis > > nick_theodora...@hotmail.com > > contact form:http://theodorakis.net/contact.html > > and for seq alignment Bioedit and MEGA 4... > > > ------------------------------ > > Message: 4 > Date: Tue, 13 Jan 2009 14:33:13 GMT > From: Aawara Chowdhury > Subject: Re: DNA software question > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain; charset=us-ascii > > In , > Han wrote: > > > Since the Mac is about to die, he would like software for a PC (XP Pro) > > that could best perform those functions. DNAStar (new version) costs > > $1,000/year in licensing costs, which colleague doesn't regard as a wise > > investment. What are his best options for free or cheap software with > > similar capabilities as what he wants of DNAStar (primer design, sequence > > alignment, etc)? > > > > We use "ApE" (A Plasmid Editor) and "Serial Cloner". > > ApE is available from: > > > Serial Cloner is available from: > > > Both are free, although a donation is suggested for Serial Cloner. > > ApE reads ABI sequencing trace files, does restriction digests, > sequence alignments, finds primers, finds silent restriction endonuclease > recognition sites, does translations, produces excellent annotated > plasmid maps, and can BLAST sequences. > > Serial Cloner does everything that ApE does, and in addition, shRNA > design, Gateway(tm) cloning design, and one can setup ligations of > 1, 2, or 3 fragments. The plasmid maps generated by Serial Cloner are > similar to those made by DNA Strider (i.e. restriction sites but not > annotated). > > AC > -- > Email: echo 36434455860060025978157675027927670979097959886449930P | dc > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 44, Issue 12 > *************************************** > -- Dr V K Gupta Sr Microbiologist (Molecular Biology) Insect Molecular Biology Lab Department of Entomology Punjab Agricultural University Ludhiana (Pb)-141004- India M: 09815963210 From blackhole from abuse.plus.com Mon Jan 19 04:45:32 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Mon Jan 19 14:28:36 2009 Subject: Low concentration miniprep References: Message-ID: <6Slj9PC8uEdJFABp@abuse.plus.com> Historians believe that in newspost on Sat, 17 Jan 2009, shifali chatrath penned the following literary masterpiece: > ?Just a query! Are satellite colonies correctly defined as slow growing colonies or a daugther colony of nearby parent colony? Neither. Satellite colonies arise on electroporated/transformed E.coli ampicillin plates when the ampicillin resistant colony's beta lactamase destroys ampicillin in the vicinity of that colony. As there is now no ampicillin in the media around that colony, background non-plasmid plated E.coli cells start to grow, giving rise to so-called satellite colonies. It doesn't happen with a kanamycin as the antibiotic is not destroyed. The use of more beta lactamase resistant antibiotics such as carbenicillin will reduce satellite formation. I still use 80:20 Methicillin:ampicillin, as I have a very old large bottle of methicillin from before Sigma stopped selling it. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From hiraghuraman from gmail.com Mon Jan 19 08:03:05 2009 From: hiraghuraman from gmail.com (Raghu) Date: Mon Jan 19 14:28:43 2009 Subject: Conversion of kda into grams Message-ID: Hi all members, A single cell of E.coli weighs 2*10 raise -12 gms- dry weight. 13% of the dry weight corresponds to proteins. one protein of molecular wt 50 kda is 0.1% of dry wt of E.coli. If this is d case then wats the mass of dat protein in gms. Plz answer this question if any1 cud solve.... From nobody from nospam.not Mon Jan 19 08:41:51 2009 From: nobody from nospam.not (Han) Date: Mon Jan 19 14:28:51 2009 Subject: Conversion of kda into grams References: Message-ID: Raghu wrote in news:bdac4aba-a797-4180-9dba- 3fd354a13cae@f40g2000pri.googlegroups.com: > Hi all members, > > A single cell of E.coli weighs 2*10 raise -12 gms- dry > weight. 13% of the dry weight corresponds to proteins. one protein of > molecular wt 50 kda is 0.1% of dry wt of E.coli. If this is d case > then wats the mass of dat protein in gms. Plz answer this question if > any1 cud solve.... > There is too much data. The MW of the protein is immaterial, and so is the % of the dry weight as total protein. 1 E coli is 2 pg, or 2000 fg 0.1% is 2 fg as the weighting of that protein per E. coli cell. -- Han email address is invalid From SBrown from ccia.unsw.edu.au Mon Jan 19 23:04:07 2009 From: SBrown from ccia.unsw.edu.au (Scott Brown) Date: Mon Jan 19 23:29:38 2009 Subject: Difficult pcr templates Message-ID: <2A67EA781EC7F949A2AB0A0D07A86C6A04C55246@mail01.ccia.local> Hi, I have recently been using clontechs GC-2 advantage pcr mix to aid in amplification of GC rich sequences, can anyone recommend or has anyone trialed any other pcr mixes on the market? Scott Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From lautys from gmail.com Tue Jan 20 04:25:48 2009 From: lautys from gmail.com (lautys) Date: Tue Jan 20 13:22:52 2009 Subject: Difficult pcr templates In-Reply-To: <2A67EA781EC7F949A2AB0A0D07A86C6A04C55246@mail01.ccia.local> References: <2A67EA781EC7F949A2AB0A0D07A86C6A04C55246@mail01.ccia.local> Message-ID: > Hi, > > I have recently been using clontechs GC-2 advantage pcr mix to aid in > amplification of GC rich sequences, can anyone recommend or has anyone > trialed any other pcr mixes on the market? > > Scott > > Hi. I'm not sure about your pcr situation, but a friend of mine is using a type of PCR mix called "failsafe PCR system" from Epicentre for her difficult PCR template as well, and she seem getting her result. The link: http://www.epibio.com/item.asp?ID=294 Good Luck. Lau From ivanoov from gmail.com Tue Jan 20 04:06:53 2009 From: ivanoov from gmail.com (chovek69) Date: Tue Jan 20 13:23:11 2009 Subject: Difficult pcr templates References: Message-ID: On Jan 20, 6:04?am, "Scott Brown" wrote: > Hi, > > I have recently been using clontechs GC-2 advantage pcr mix to aid in > amplification of GC rich sequences, can anyone recommend or has anyone > trialed any other pcr mixes on the market? > > Scott > > Scott Brown > PhD Candidate > Molecular Carcinogenesis Program > Children's Cancer Institute of Australia for Medical Research > High Street (PO Box 81) > RANDWICK NSW 2031 > AUSTRALIA > > Phone: +61 2 9382 1829 > Fax: +61 2 9382 1850 > > Email: sbr...@ccia.unsw.edu.au > Web:www.ccia.org.au > > Children's Cancer Institute Australia is the only independent medical See this... helps alot with difficult templates... www.molgen.mpg.de/~ag_krobitsch/16842759.pdf > research institute in Australia solely devoted to research into the > causes, prevention and cure of childhood cancer. Our vision is to save > the lives of all children with cancer and eliminate their suffering. > > The information contained in this message and any annexure is > confidential and intended only for the named recipient(s). If you have > received this message in error, please contact the sender immediately > and destroy the original message. > > Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. > > The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. From stewjw from gmail.com Tue Jan 20 05:11:04 2009 From: stewjw from gmail.com (StewJW) Date: Tue Jan 20 13:23:16 2009 Subject: pI determination methods for QC References: <8Xycl.26928$Nq5.13176@newsfe24.iad> Message-ID: Invitrogen do offer the precast Novex IEF gels which are used in their standard Surelock appararatus but they range from pH 3-10 or pH 3-7. For crude estimation there are also IEF markers in the 3.5 - 10.7 range. The IPG (immobiliased pH gradient strips) Runner system you mentioned is designed for running 2D gels of a large number of samples. There is a gel strip holder, IPGRunner Cassette, which fits inside the Invitrogen Surelock mini-gel apparatus. Each cassette can hold upto 6 (7cm) strips. The strips come in the following pH ranges: ZOOM? Strips pH 3-10NL 12 ZM0011 ZOOM? Strips pH 4-7 12 ZM0012 ZOOM? Strips pH 6-10 12 ZM0013 ZOOM? Strips pH 4.5-5.5 12 ZM0014 ZOOM? Strips pH 5.3-6.3 12 ZM0015 ZOOM? Strips pH 6.1-7.1 After running the IEF strips they are individually removed and placed long ways inside an insert of a precast ZOOM? Gels, which are are 8 x 8 cm, 1.0 mm thick pre-cast polyacrylamide gels to run the second dimension. http://tools.invitrogen.com/Content/SFS/ProductNotes/F_071215_ZOOM%20IPG%20Runner-MKT-RD-TL-HL.pdf http://tools.invitrogen.com/content/sfs/manuals/zoomipgrunner_man.pdf I've never used this, and I'm unsure how practical it is to excise small samples from gels to measure their pH in solution. Thermo- Russell sells a small Ag/AgCl combination electrode which can measure down to 50?l of liquid, catalogue number: CMAW711/4/5/SC; dimensions below cap are 90mm x 5.5mm. See www.thermo.com/russell. I should say I work for Fisher UK a ThermoFisher company so I'm a little biased in my recommendation. From stewjw from gmail.com Tue Jan 20 06:01:17 2009 From: stewjw from gmail.com (StewJW) Date: Tue Jan 20 13:23:21 2009 Subject: Difficult pcr templates References: Message-ID: Thanks for the previous post, interesting paper. A possible commercial solution, as well a the PCRX Enhancer System mentioned in the paper, is AccuPrime=99 GC-Rich DNA Polymerase, I've no idea why this polymerase should work any better: http://tools.invitrogen.com/content/sfs/manuals/12337.pdf From C.P.Pena-Diaz from 2006.hull.ac.uk Tue Jan 20 11:21:18 2009 From: C.P.Pena-Diaz from 2006.hull.ac.uk (Carmen P Pena Diaz) Date: Tue Jan 20 13:23:30 2009 Subject: Difficult pcr templates References: <2A67EA781EC7F949A2AB0A0D07A86C6A04C55246@mail01.ccia.local> Message-ID: <445800D8FD5D3D43878FD7D2842A3047435414@EXCL2VS2.adir.hull.ac.uk> Phusion polymerase & 5x GC buffer altogether (from Finnzymes) works wonders.... good luck P. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of Scott Brown Sent: Tue 20/01/2009 04:04 To: methods@magpie.bio.indiana.edu Subject: Difficult pcr templates Hi, I have recently been using clontechs GC-2 advantage pcr mix to aid in amplification of GC rich sequences, can anyone recommend or has anyone trialed any other pcr mixes on the market? Scott Scott Brown PhD Candidate Molecular Carcinogenesis Program Children's Cancer Institute of Australia for Medical Research High Street (PO Box 81) RANDWICK NSW 2031 AUSTRALIA Phone: +61 2 9382 1829 Fax: +61 2 9382 1850 Email: sbrown@ccia.unsw.edu.au Web: www.ccia.org.au Children's Cancer Institute Australia is the only independent medical research institute in Australia solely devoted to research into the causes, prevention and cure of childhood cancer. Our vision is to save the lives of all children with cancer and eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error, please contact the sender immediately and destroy the original message. Children's Cancer Institute Australia for Medical Research is the only independent medical research institute in Australia devoted to research into the causes, prevention, better treatment and ultimately a cure of childhood cancer. Our vision is to save the lives of all children with cancer and to eliminate their suffering. The information contained in this message and any annexure is confidential and intended only for the named recipient(s). If you have received this message in error please contact the sender immediately and destroy the original message. _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods -------------- next part -------------- ***************************************************************************************** To view the terms under which this email is distributed, please go to http://www.hull.ac.uk/legal/email_disclaimer.html ***************************************************************************************** From Nikola.Wenta from nottingham.ac.uk Tue Jan 20 12:45:27 2009 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Tue Jan 20 13:23:35 2009 Subject: Conversion of kda into grams In-Reply-To: <200901201703.n0KH3j814407@net.bio.net> References: <200901201703.n0KH3j814407@net.bio.net> Message-ID: <814A9FCB0AD790489679BFBCE6CA5E55B1198B@VUIEXCHC.ad.nottingham.ac.uk> Hi Raghu, 1 kDa corresponds to 1000 Dalton; 1 Dalton is 1/12 of the molecular mass of the C12 isotope. 1 Dalton therefore is 1.66053878E-24 g. Your 50 kDa protein should therefore have 8.3026939E-23 g. If you take this times 1000 (0.1%), then you have 8.3026939E-19 g of your protein in that cell. This is equal to 8.3E-4 fg. If you don't mind, please try to improve your expression. BCause otherwize no1 can undastand ya. And as this is a scientific forum ( NOT A CHAT OR AN SMS ), you should try to speak like a scientist, right? By the way, even if I don't recommend Wikipedia as a reliable source of knowledge, you might have a look at: http://de.wikipedia.org/wiki/Dalton Best wishes Niko -----Original Message----- Subject: Conversion of kda into grams To: methods@net.bio.net Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Hi all members, A single cell of E.coli weighs 2*10 raise -12 gms- dry weight. 13% of the dry weight corresponds to proteins. one protein of molecular wt 50 kda is 0.1% of dry wt of E.coli. If this is d case then wats the mass of dat protein in gms. Plz answer this question if any1 cud solve.... This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation. From holeung from berkeley.edu Wed Jan 21 15:48:17 2009 From: holeung from berkeley.edu (Ho-Leung Ng) Date: Wed Jan 21 16:52:16 2009 Subject: Difficult pcr templates Message-ID: <6678762a0901211248n3e1ed29dlde4159645198c595@mail.gmail.com> For the toughest templates, I've had success with Stratagene Herculase II. Of course, also try DMSO or betaine with your enzyme of choice. ho UC Berkeley From shifalich from rediffmail.com Wed Jan 21 23:50:30 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Jan 22 12:33:29 2009 Subject: Conversion of kda into grams Message-ID: <20090122045030.35618.qmail@f4mail-235-231.rediffmail.com> I also agree with the answer below. MW is immaterial, 2fg is the answer! Shifali ? On Mon, 19 Jan 2009 Han wrote : >Raghu wrote in news:bdac4aba-a797-4180-9dba- >3fd354a13cae@f40g2000pri.googlegroups.com: > > > Hi all members, > > > > A single cell of E.coli weighs 2*10 raise -12 gms- dry > > weight. 13% of the dry weight corresponds to proteins. one protein of > > molecular wt 50 kda is 0.1% of dry wt of E.coli. If this is d case > > then wats the mass of dat protein in gms. Plz answer this question if > > any1 cud solve.... > > >There is too much data. The MW of the protein is immaterial, and so is the >% of the dry weight as total protein. >1 E coli is 2 pg, or 2000 fg >0.1% is 2 fg as the weighting of that protein per E. coli cell. > >-- >Han >email address is invalid >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From m.saidi from cnstn.rnrt.tn Thu Jan 22 07:42:35 2009 From: m.saidi from cnstn.rnrt.tn (Mouldi SAIDI) Date: Thu Jan 22 12:34:06 2009 Subject: (pas de sujet) Message-ID: <497869BB.4040808@cnstn.rnrt.tn> Dear all, I would like to ask if the separation of casein using HPLC, C18 RP and acetonitrile/water TFA is possible. I'm afraid that acetonitrile precipitates proteins? Best regards From derek_riley_stein from hotmail.com Thu Jan 22 18:10:27 2009 From: derek_riley_stein from hotmail.com (Derek Stein) Date: Thu Jan 22 19:17:58 2009 Subject: Ammonium Formate Message-ID: =20 Hi=2C =20 How would I go about making a 10mM ammonium formate=2CpH3=2C 25% ACN buffer= and then a 500mM ammonium formate=2CpH6.8=2C 25% ACN buffer. I want to ju= st weigh out the crystal form ammonium formate and then adjust the pH. But= this may be wrong. Someone has told me to start with formic acid and adju= st the pH with ammonium-OH. This is all well an good for the first solutio= n but the second solution would (500mM) have way to much formic acid in it = for me to get the pH up to 6.8. Not sure how to properly make each of thes= e buffers. =20 Derek Derek Stein Masters Student 507-745 Bannatyne Avenue Department of Medical = Microbiology University of Manitoba Winnipeg=2C Manitoba R3E 0J9Ph: (204) 7= 89-3202 Cell: (204) 290-5996 Web Site: http://www.mrsi.ca _________________________________________________________________ Drag n=92 drop=97Get easy photo sharing with Windows Live=99 Photos. http://www.microsoft.com/windows/windowslive/photos.aspx= From shifalich from rediffmail.com Fri Jan 23 04:59:34 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Fri Jan 23 13:13:03 2009 Subject: Low concentration miniprep Message-ID: <20090123095934.11802.qmail@f4mail204.rediffmail.com> ? Dear all! Thankyou very much for prividing me wealthy info on satellite colonies. Shifali On Mon, 19 Jan 2009 Duncan Clark wrote : >Historians believe that in newspost on Sat, 17 Jan 2009, shifali chatrath penned the following literary masterpiece: >> ?Just a query! Are satellite colonies correctly defined as slow growing colonies or a daugther colony of nearby parent colony? > > >Neither. > >Satellite colonies arise on electroporated/transformed E.coli ampicillin plates when the ampicillin resistant colony's beta lactamase destroys ampicillin in the vicinity of that colony. As there is now no ampicillin in the media around that colony, background non-plasmid plated E.coli cells start to grow, giving rise to so-called satellite colonies. > >It doesn't happen with a kanamycin as the antibiotic is not destroyed. > >The use of more beta lactamase resistant antibiotics such as carbenicillin will reduce satellite formation. I still use 80:20 Methicillin:ampicillin, as I have a very old large bottle of methicillin from before Sigma stopped selling it. > >Duncan >-- I love deadlines. I especially like the whooshing noise they make as >they go flying by. > >Duncan Clark >GeneSys Ltd. >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From shifalich from rediffmail.com Fri Jan 23 05:02:48 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Fri Jan 23 13:14:50 2009 Subject: (pas de sujet) Message-ID: <20090123100248.48349.qmail@f4mail-234-122.rediffmail.com> ? We regularly purify proteins on RPHPLC C18 column. I have never heard of any protein getting pptd. with ACN. I suggest you to go ahed with your purification! All the best! On Thu, 22 Jan 2009 Mouldi SAIDI wrote : >Dear all, >I would like to ask if the separation of casein using HPLC, C18 RP and acetonitrile/water TFA is possible. >I'm afraid that acetonitrile precipitates proteins? >Best regards > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From engelbert_buxbaum from hotmail.com Fri Jan 23 13:15:03 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jan 23 16:03:27 2009 Subject: Ammonium Formate References: Message-ID: Am 22.01.2009, 19:10 Uhr, schrieb Derek Stein : > How would I go about making a 10mM ammonium formate,pH3, 25% ACN buffer > and then a 500mM ammonium formate,pH6.8, 25% ACN buffer. I want to just > weigh out the crystal form ammonium formate and then adjust the pH. But > this may be wrong. Someone has told me to start with formic acid and > adjust the pH with ammonium-OH. This is all well an good for the first > solution but the second solution would (500mM) have way to much formic > acid in it for me to get the pH up to 6.8. Not sure how to properly > make each of these buffers. For a purely aqueous solution you need a mixture of formic acid and ammonium formate, the ratio calculated from the Henderson-Hasselbalch-equation, log([A]/[HA]) + pKa = pH Example: Your pH is 3.0, the pKa of formic acid is 3.75. Hence log([A]/[HA]) = 3 - 3.75 = -0.75 and [A]/[HA] = antilog(-0.75) = 0.178. At the same time your total concentration [A]+[HA] = 10 mM. Hence 10 mM = 1.178 [HA] and [HA] = 8.5 mM. So you need to prepare a solution of 8.5 mM formic acid and 10 mM - 8.5 mM = 1.5 mM ammonium formate. It is probably easiest to prepare that as a concentrate, say, 10-fold. The molecular mass of ammonium formate is 63.06, hence you need 946 mg/l. The formic acid is best added from a solution of known (from titration) concentration. If that solution were 1 M, you'd need 85 ml/l. Note however that adding organic solvent changes the activity of the ions and hence the pH. Whether or not the original author has corrected for that effect is a different question. If the paper was written properly (giving all the info necessary to repeat the experiment) it should state this. The other problem with your question is that formate has essentially no buffering capacity at pH 6.8. Buffers work only within +/- 1 pH-unit of their pKa, hence you would use formate only between about 3 and 5. Neither would the ammonium ion (pKa = 9.2) have much buffering capacity at this pH. One further aspect: Ammonium formate is often used as volatile buffer, that vanishes upon lyophilisation. In my experience however in this situation the water and ammonia evaporate first, leaving the sample in concentrated formic acid, which may well melt due to its relatively low vapor pressure. Whether or not the sample survives that depends on its chemistry. Ammonium bicarbonate is truly volatile and buffers well in the pH 7-9 range. From swamyarul from gmail.com Fri Jan 23 23:02:53 2009 From: swamyarul from gmail.com (Arul Narayanasamy) Date: Fri Jan 23 23:42:26 2009 Subject: Doubt regd about packing a column Message-ID: Dear Sir/Mam Well.Kind greetings.My name is Arul doing PhD IN south Korea in the field of cancer bio marker.I have a doubt about packing a AAL(Lectin bed) to pierce centrifuge column,5ml,product number-89897.Kindly let me know how to pack the AAL bed in to this column.I would grateful to you if give a suggestion regarding this.I am looking forward to hear from you. Thanks and regards Arul From prae from gmx.net Sat Jan 24 16:49:37 2009 From: prae from gmx.net (Christian Praetorius) Date: Sun Jan 25 12:19:50 2009 Subject: Low concentration miniprep References: <6Slj9PC8uEdJFABp@abuse.plus.com> Message-ID: <6u1gnhFcaahfU2@mid.individual.net> Duncan Clark wrote: >The use of more beta lactamase resistant antibiotics such as >carbenicillin will reduce satellite formation. I still use 80:20 >Methicillin:ampicillin, as I have a very old large bottle of methicillin >from before Sigma stopped selling it. Are cells carrying a ampicillin resistence plasmid resistent to carbenicillin/methicillin? Christian -- X-no-Sig: yes From gsridhar01 from gmail.com Sun Jan 25 04:25:28 2009 From: gsridhar01 from gmail.com (sridhar gunday) Date: Sun Jan 25 12:20:05 2009 Subject: enzyme assy stadradigation Message-ID: <4815f10a0901250125v426efb76v6addbc9f9e9ff80d@mail.gmail.com> I am doing IDO enzyme asssy , for this according the litrature it was 200ul. s but idon't have that much of small volume cuivett that's way i was increased the total reaction mixtue , is it correct procedurei am foolowing -specilly i am tacking the results with spectrofluoremeter -- G.Sridhar C/o Prof.T.Naga Raju, Dept.of Zoology, university college of science, osmania university, Hyderabad-07,India From aawara from pontiff-playground.org Sun Jan 25 10:37:26 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Sun Jan 25 12:20:23 2009 Subject: Low concentration miniprep References: <6Slj9PC8uEdJFABp@abuse.plus.com> <6u1gnhFcaahfU2@mid.individual.net> Message-ID: In <6u1gnhFcaahfU2@mid.individual.net>, Christian Praetorius wrote: > Duncan Clark wrote: > >>The use of more beta lactamase resistant antibiotics such as >>carbenicillin will reduce satellite formation. I still use 80:20 >>Methicillin:ampicillin, as I have a very old large bottle of methicillin >>from before Sigma stopped selling it. > > Are cells carrying a ampicillin resistence plasmid resistent to > carbenicillin/methicillin? DH5a carrying pBR322/pMB15-based low-copy plasmids are resistant to carbenicillin (at 100 ug/ml). I prefer carbenicillin to ampicillin because it is more resistant to degradation by secreted beta-lactamase, and significantly reduces the number of satellite colonies. Also, our carbenicillin plates stay good in the cold room for over 6 months. We have also used Timentin for the same purpose (it is a mixture of a beta-lactam antibiotic - ticarcillin, and the beta-lactamase i nhibitor clavulonic acid). Somewhat difficult to obtain without a prescription these days. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From yarriofultramar from gmail.com Mon Jan 26 06:28:24 2009 From: yarriofultramar from gmail.com (Yarri) Date: Mon Jan 26 12:27:54 2009 Subject: 5' RACE troubleshooting Message-ID: <2f4c0f41-827d-457d-84b2-f4b16c3293c3@p2g2000prf.googlegroups.com> Hej! I am using a SMART RACE cDNA Amplification Kit from Clontech in order to get cDNA from roots of Datisca glomerata. I have been able to get few clones from cDNA I amplified with primers that had high Tm values - as recommended by the protocol. Unfortunately, now I have to work with more troublesome sequences, where best primers that can be designed have a Tm value <64C. I was trying to follow the protocol without much success. Does anyone has some experience with this kit or some suggestions how to approach the problem? I am new to RACE so I am not exactly sure how to troubleshoot RACE conditions. As an example: A sequencing result: CATGGAGGATGAGAAACAATATTACTAAAATAACTTTATGTTATGTGCTGTTTGAAGTTTATTTCTCTATGT TATAACTGCATGCTGCTATCGATCATTGTGAAACAAATCCTTATCAAATATTAATTATATATCTAGCCTTCT GACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAMAAAAACAAA AAAACAAAACAAACAA GSP2 primer I have designed: CACAATGATCGATAGCAGCATGCAG NSP2 primer: GTAATATTGTTTCTCATCCTCCATG Now I am waiting for additional primer that can be used as a nested one: GTAATATTGTTTCTCATCCTCCATG Thanks! Marian P?aszczyca From J-R.Kraehling from tu-braunschweig.de Mon Jan 26 10:01:35 2009 From: J-R.Kraehling from tu-braunschweig.de (=?iso-8859-1?Q?Jan_R._Kr=E4hling?=) Date: Mon Jan 26 12:28:04 2009 Subject: Ultrasound-mediated gene transfer Message-ID: <37DC72C79C2543C49237499FC4C8A5F6@Jan> Dear all, I have read a most interesting article on ultrasound-mediated gene transfer "Sonoporation" (Fischer et al. 2006 Journal of Biotechnology 122, 393-411.) It seems that you can use a standard laboratory sonicator commonly used to disrupt cells to transfect cells with expression plasmids. Seemingly this method works especially well with neuronal cells or related cells such as PC-12. Does anyone have practical experience or a good protocol in addition to the one described by Fischer? For me it seems too good to be true that you can replace the expensive transfection reagents by this simple methods. Is it in your opinion worth to try? Thanks for your kind help Jan From aawara from pontiff-playground.org Mon Jan 26 10:34:16 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Mon Jan 26 12:28:15 2009 Subject: Low concentration miniprep References: <6Slj9PC8uEdJFABp@abuse.plus.com> <6u1gnhFcaahfU2@mid.individual.net> Message-ID: In , DK wrote: > In article , aawara@pontiff-playground.org wrote: >>We have also used Timentin for the same purpose (it is a mixture of >>a beta-lactam antibiotic - ticarcillin, and the beta-lactamase i >>nhibitor clavulonic acid). Somewhat difficult to obtain without a >>prescription these days. > > No prescription and reasonably priced: > http://www.goldbio.com/details.php?productId=266&categoryId=4 Nice. When I was a graduate student (20 years ago), I used to just walk over to the pharmacy at the hospital attached to CWRU, pick up a couple bottles of Timentin, and have it charged to my mentor's grant. Now, I have to get one of my colleagues who's an MD to procure it for us (on the rare occasion when we're expressing a protein that gives very low yield). But it is nice to know that the same combination of drugs is available without going through this rigmarole. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From editor from gene-quantification.info Mon Jan 26 03:27:18 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Mon Jan 26 12:48:36 2009 Subject: qPCR NEWS January 2009 - Lab-on-Chip special edition Message-ID: qPCR NEWS January 2009 - Lab-on-Chip special edition ---------------------------------------------------------------------------= ----- Dear researcher, dear Gene Quantification page reader, Our newsletter informs about the latest news in quantitative real-time PCR (qPCR and qRT-PCR), which are compiled and summarised on the Gene Quantification homepage. The focus of this newsletter issue is: - qPCR 2009 Event =3D> http://www.qPCR2009.net - we are proud to present 230 scientific contributions (86 talks & 144 posters) have a look on the preliminary online agenda - http://online-AGENDA.qpcr2009.net/ - Lab on Chip application papers - PCR efficiency page updated with the newest papers in the field - Online translation service of the Gene Quantification =3D> http://translation.gene-quantification.info/ - European wide qPCR application workshops =3D> register now ! =3D> qPCR course program spring - summer 2009 =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f ---------------------------------------------------------------------------= ----- Lab-on-a-chip (LOC) is a term for devices that integrate (multiple) laboratory functions on a single chip of only millimeters to a few square centimeters in size and that are capable of handling extremely small fluid volumes down to less than pico liters. Lab-on-a-chip devices are a subset of MEMS devices and often indicated by "Micro Total Analysis Systems" (=B5TAS) as well. Microfluidics is a broader term that describes also mechanical flow control devices like pumps and valves or sensors like flowmeters and viscometers. However, strictly regarded "Lab-on-a-Chip" indicates generally the scaling of single or multiple lab processes down to chip-format, whereas "=B5TAS" is dedicated to the integration of the total sequence of lab processes to perform chemical analysis. The term "Lab-on-a-Chip" was introduced later on when it turned out that =B5TAS technologies were more widely applicable than only for analysis purposes. Microfluidics deals with the behavior, precise control and manipulation of fluids that are geometrically constrained to a small, typically sub-milimeter, scale. It is a multidisciplinary field intersecting engineering, physics, chemistry, microtechnology and biotechnology, with practical applications to the design of systems in which such small volumes of fluids will be used. Microfluidics has emerged in the beginning of the 1980s and is used in the development of inkjet printheads, DNA chips, lab-on-a-chip technology, micro- propulsion, and micro-thermal technologies. http://lab-on-chip.gene-quantification.info/ ---------------------------------------------------------------------------= ----- - Nanodroplet real-time PCR system with laser assisted heating - A polymer lab-on-a-chip for reverse transcription (RT)-PCR based point-of-care clinical diagnostics - Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification - PCR thermal management in an integrated Lab on Chip - Rapid PCR in a continuous flow device - Single-molecule DNA amplification and analysis in an integrated microfluidic device - Microfabricated PCR-electrochemical device for simultaneous DNA amplification and detection - Miniaturized flow-through PCR with different template types in a silicon chip thermocycler - Coordinating Multiple Droplets in Planar Array Digital Microfluidic Systems - Microcontact Printing of DNA Molecules - Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes - ITO-coated glass/polydimethylsiloxane continuous-flow PCR chip - and much more here http://lab-on-chip.gene-quantification.info/ ---------------------------------------------------------------------------= ----- LOC technology for total RNA and microRNA quality & quantity control - Bioanlyzer 2100 - Experion - RNA integrity measurements - RIN and RQI algorithms - find more information and papers here =3D> http://rna-integrity.gene-quantification.info/ ---------------------------------------------------------------------------= ----- online translation Since October 2008 we provide an online translation service of the Gene Quantification pages to several languages. Please recognize this is an automatic and robotic based translation service, and therefore we can give NO guarantee about the generated content. It should help to understand the "rough" content of the Gene Quantification pages, but still the original is the ENGLISH version: http://translation.gene-quantification.info/ http://www.gene-quantification.asia http://chinese.gene-quantification.asia/ http://dutch.gene-quantification.info/ http://french.gene-quantification.info/ http://german.gene-quantification.info/ http://greek.gene-quantification.info/ http://italian.gene-quantification.info/ http://japanese.gene-quantification.asia/ http://korean.gene-quantification.asia/ http://portuguese.gene-quantification.info/ http://russian.gene-quantification.asia/ http://spanish.gene-quantification.info/ ---------------------------------------------------------------------------= ----- With the new qPCR INFO PORTAL and all the presented tools we will help you with to find the right information about qPCR and related topics in Molecular Biology in the literature and in the World Wide Web. =3D> Papers / Protocols / Methods / Databases / Alets / Feeds / Books / Forums / E-mail / Directory http://infoportal.gene-quantification.info/ ---------------------------------------------------------------------------= ----- Upcoming Events World-wide academic and commercial qPCR Events http://events.gene-quantification.info/ Symposia, Meetings, Conferences, Workshops, Seminars, Online-Seminars, qPCR Education Program, etc. Please submit your qPCR event here =3D> events@gene- quantification.info ---------------------------------------------------------------------------= ----- qPCR 2009 EVENT - 9 - 13 March 2009 more news here =3D> http://www.qPCR2009.net download event FLYER =3D> http://www.bioeps.com/qpcr2009/qPCR-2009-3rd-an= nouncement.pdf We are proud to present 230 scientific contributions (86 talks & 144 posters) have a look on the preliminary online agenda =3D> http://online-AGENDA.qpcr2009.net/ It is a pleasure to announce the Nobel Prize Laureate Kary Mullis at the qPCR 2009 event in an own session =9325th Anniversary of PCR=94 List of confirmed speakers =3D> http://speakers.qpcr2009.net/ An industrial exhibition with the 30 world leading companies will be held during the qPCR Symposium March 9-11 =3D> http://exhibition.qpcr2009= .net/ Our sponsors =3D> http://sponsors.qpcr2009.net/ Please register until 31st December to get the early registartion fee =3D> http://registration.qpcr2009.net/ Keynote lectures The Pioneer in PCR Kary B. Mullis 1993 Nobel Prize in Chemistry - 25th Anniversary of PCR Stephen A. Bustin Professor of Molecular Science, Institute of Cell and Molecular Science, Queen Mary's School of Medicine and Dentistry, University of London, UK - A new qPCR assay for the detection of Clostridium difficile - MIQE- guidelines for publication of qPCR data Ken Livak Senior Scientific Fellow, Fluidigm Corporation,San Francisco, CA, US - Moving from qPCR Assays to qPCR Arrays. ---------------------------------------------------------------------------= ----- qPCR WORKSHOP BioEPS GmbH / TATAA Biocenter Germany - qPCR Application workshops At the TATAA Biocenter Germany we offer qPCR application workshops, a 3-day qPCR Core Module and a 2-day qPCR Biostatistics Module. All courses are held regularly in G=F6teborg, Sweden, in English and in Freising-Weihenstephan, Germany, in German and English, and in Prague, Czech Republic in English and Czech. Depending on the occasion the workshop language and the different prices may apply. Further customized workshops and specialized trainings will be held as well across Europe and world-wide. TATAA Biocenter Germany workshops are held in cooperation with BioEPS GmbH, located at the campus of the Technical University of Munich, in Freising-Weihenstephan, very close to the Munich Airport (MUC). For more information and registration, please see our web page: =3D> http://TATAA.gene-quantification.info/ Course Occasions 2009: 3-day qPCR Core Module (Mon. - Wed.) 2-day BioStatistics Module (Thu. - Fri.) 3-day single-cell qPCR Module (Mon. - Wed.) 3-day microRNA Module (Mon. - Wed.) 26 - 30th January 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 20 - 22 April 2009 (E) NEW microRNA qPCR 20 - 22 April 2009 (E) NEW singel-cell qPCR Application Workshop 11 - 15 May 2009 (Deutsch) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 15 - 19 Juni 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) 13 - 15 Juli 2009 (E) NEW microRNA qPCR 27 - 31 Juli 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2-day BioStatistics Module (Thu. - Fri.) =3D> http://www.gene-quantification.de/bioeps-courses-spring-summer-2009.pd= f ---------------------------------------------------------------------------= ----- Forward Please send the qPCR NEWS to further scientists and friends who are interested in qPCR ! Best regards, Michael W. Pfaffl responsible Editor of the Gene Quantification Pages http://www.gene-quantification.info ---------------------------------------------------------------------------= ----- If this newsletter is not displayed correctly by your email client, please use following link: http://qpcrnews.gene-quantification.info/ The qPCR NEWS and the Gene Quantification Pages are educational sites with the only purpose of facilitating access to qPCR related information on the internet. The qPCR NEWS and the Gene Quantification Pages are edited by Michael W. Pfaffl. Copyright =A9 2005 - 2009 All rights reserved. Any unauthorized use, reproduction, or transfer of this message or its contents, in any medium, is strictly prohibited. Disclaimer & Copyrights are displayed on the homepage www.gene-quantification.com To subscribe or change your e-mail address in qPCR NEWS, and if you would like to receive future issues FREE of charge, please send an e- mail with the subject SUBSCRIBE to mailto:newsletter@gene- quantification.info?subject=3DSUBSCRIBE From blackhole from abuse.plus.com Mon Jan 26 12:40:29 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue Jan 27 12:32:17 2009 Subject: Low concentration miniprep References: <6Slj9PC8uEdJFABp@abuse.plus.com> <6u1gnhFcaahfU2@mid.individual.net> Message-ID: Historians believe that in newspost on Sun, 25 Jan 2009, DK penned the following literary masterpiece: >In article , aawara@pontiff-playground.org wrote: >>We have also used Timentin for the same purpose (it is a mixture of >>a beta-lactam antibiotic - ticarcillin, and the beta-lactamase i >>nhibitor clavulonic acid). Somewhat difficult to obtain without a >>prescription these days. > >No prescription and reasonably priced: >http://www.goldbio.com/details.php?productId=266&categoryId=4 > >DK You haven't come across any readily available sources of Methicillin have you? I'll be honest, I haven't looked for some time. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From jxx from ou.edu Tue Jan 27 17:59:30 2009 From: jxx from ou.edu (Jiang, Xiaoxu) Date: Tue Jan 27 18:28:15 2009 Subject: filter for heme Message-ID: <244D7364B94F274E9786B58135DED6490144194D24F2@XMAIL5.sooner.net.ou.edu> Dear all, I am a PhD student. I am working on a isotope labeled heme uptake experiment by bacteria cells. Now I have a problem about the filter for heme: after I incubate my cells with heme in medium, I am trying to filter out the unbound heme and take the membrane filter to the gamma counter with the cells on it. But I find there are always a lot of unbounded heme sticks to the membrane and generates a high background, which pretty much ruins the experiments. I have tried several different kinds of membrane filters and right now I am using celluloseacetat filter but the background is still quite high. I want to ask that what kind of membrane filter has the least non-specific binding for heme? And also, what kind of washing buffer is good to wash the filter to get rid of the non specific binding heme. Thank you! From pdeitik from bcm.tmc.edu Tue Jan 27 18:50:37 2009 From: pdeitik from bcm.tmc.edu (Deitiker, Philip R) Date: Wed Jan 28 00:03:58 2009 Subject: filter for heme In-Reply-To: <244D7364B94F274E9786B58135DED6490144194D24F2@XMAIL5.sooner.net.ou.edu> Message-ID: Heme likes to interact with polymers and filters are composed of polymers. Although I was never successful at recoving DNA from heparanized cells, I was able to recover cells from extracellular, 'lysed cell', heme by spinning them across a glycerol step that separated heme bound material from cells. If I recall correctly one needs 8 to 10% glycerol to create a barrier robust enough to keep aggregate heme bound material afloat, this was with whole heparinized blood in RBC lysis buffer. One can carefully layer the glycerol under the suspended cells and centrifuge them. For the most part the cells recovered only had heme bound on or within the cells. The DNA yeild was low, the iron apparently gets to the DNA first. Heparin can apparently strip the iron off of the heme group, its bad news for DNA obtained from frozen cells, cause they are almost always permeated enough to let the heparin. Since your cell suspension is probably less dense than blood you probably could get away with a lower percentage of glycerol and I would recommend removing the cells and spinning them over the step gradient in a fresh tube at least once more. One trivial observation, when heparin is present with heme, the RBC membranes precipitate and will layer at about 10% glycerol concentration in RBC lysis buffer and the membranes will be lightly coated with heme. Its possible an amalgamation of polymers (RBC RNA, protein, etc) heparin, heme and unbound iron. This indicates that heme or iron outside of the hemoglobin molecule or in the presence of heparin is quite capable of crosslinking to other materials. apomyoglobin of course binds heme so . . . . . . -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Jiang, Xiaoxu Sent: Tuesday, January 27, 2009 5:00 PM To: methods@net.bio.net Subject: filter for heme Dear all, I am a PhD student. I am working on a isotope labeled heme uptake experiment by bacteria cells. Now I have a problem about the filter for heme: after I incubate my cells with heme in medium, I am trying to filter out the unbound heme and take the membrane filter to the gamma counter with the cells on it. But I find there are always a lot of unbounded heme sticks to the membrane and generates a high background, which pretty much ruins the experiments. I have tried several different kinds of membrane filters and right now I am using celluloseacetat filter but the background is still quite high. I want to ask that what kind of membrane filter has the least non-specific binding for heme? And also, what kind of washing buffer is good to wash the filter to get rid of the non specific binding heme. Thank you! _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From eenigoy from gmail.com Wed Jan 28 01:15:16 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Wed Jan 28 09:29:43 2009 Subject: Electroporation Message-ID: <77ddf6c70901272215x2f1c9aceo9bfcd3224c08341d@mail.gmail.com> Dear All, I want to use biorad gene pulser for transforming *E. coli* with my ligated mix. It is mentioned in the bio rad manual that we must incubate the ligated mix at 65deg C for 15 min to inactivate the enzyme and then proceed with its dilution or etOH precipitation to get rid of extra salts that may decrease the resistance and cause arcing. I did go for the boiling step, but I'm afraid I'll lose the DNA by precipitation since already only about 80 to 100 ng, insert + vector DNA is present in my ligated mix. Cannot afford to lose any of it during precipitation What could be done to get rid of the salt content that hampers the electroporation? -- -- Yoginee Budhkar Junior Research Fellow Food Engineering and Technology Department Institute of Chemical Technology Matunga, Mumbai 400019 Contact: +91 9223355677 From shifalich from rediffmail.com Wed Jan 28 02:00:21 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Wed Jan 28 09:30:09 2009 Subject: 5' RACE troubleshooting Message-ID: <20090128070021.34083.qmail@f4mail-235-149.rediffmail.com> ?HI! I did not look into your primers but I just wanted to ask If you have tried with 64C as annealing temp. I know that they ask you to design primers with Tm >/= 70 but you can still try first 7 cycles with 62 and rest 32 with 65 or so. I had the same problems but even 60-65C gave me specific bands. Another way out is, you can add extra GC to 5' of your primers. I think adding 3-4 G/C will help. Just keep it in mind while reading your sequencing. Both things work well for me! HOpe it helps. All the best! Shifali On Mon, 26 Jan 2009 Yarri wrote : >Hej! > >I am using a SMART RACE cDNA Amplification Kit from Clontech in order >to get cDNA from roots of Datisca glomerata. I have been able to get >few clones from cDNA I amplified with primers that had high Tm values >- as recommended by the protocol. Unfortunately, now I have to work >with more troublesome sequences, where best primers that can be >designed have a Tm value <64C. I was trying to follow the protocol >without much success. Does anyone has some experience with this kit or >some suggestions how to approach the problem? I am new to RACE so I am >not exactly sure how to troubleshoot RACE conditions. >As an example: >A sequencing result: >CATGGAGGATGAGAAACAATATTACTAAAATAACTTTATGTTATGTGCTGTTTGAAGTTTATTTCTCTATGT >TATAACTGCATGCTGCTATCGATCATTGTGAAACAAATCCTTATCAAATATTAATTATATATCTAGCCTTCT >GACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAMAAAAACAAA >AAAACAAAACAAACAA > >GSP2 primer I have designed: CACAATGATCGATAGCAGCATGCAG >NSP2 primer: GTAATATTGTTTCTCATCCTCCATG >Now I am waiting for additional primer that can be used as a nested >one: GTAATATTGTTTCTCATCCTCCATG >Thanks! > > Marian >P?aszczyca >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods Shifali Chatrath Graduate Student Protein science Lab Dept. of Biological sciences National University of Singapore Singapore +65-65161210 +65-96393449 From novalidaddress from nurfuerspam.de Wed Jan 28 07:09:32 2009 From: novalidaddress from nurfuerspam.de (WS) Date: Wed Jan 28 09:30:53 2009 Subject: filter for heme References: Message-ID: <18154a1a-137f-4bfe-b781-bdb490fd7c2d@g1g2000pra.googlegroups.com> Dear Philip, dear Xiaoxu, instead of glycerol, you might consider mixtures of dodecane / bromododecane (for ratios, please search PubMed or Google). Pipet the cell suspension after labeling on the organic layer and briefly spin. You then may freeze the whole vial (in liquid nitrogen eg) and cut off the lower part for counting. Wo From eric.meltzer from gmail.com Wed Jan 28 09:05:54 2009 From: eric.meltzer from gmail.com (Eric Meltzer) Date: Wed Jan 28 09:31:00 2009 Subject: Common integration loci for yeast? Message-ID: I'm looking to integrate a series of reasonably large constructs into a yeast strain. I know that people commonly integrate into HO, and of course various metabolic loci like HIS or URA. In trying to figure out the best place to integrate, I've been trying to see if there are some other common loci that don't affect growth much (or only affect growth on selective media like HIS etc.). If anyone can point me in the direction of a good paper, or just a few good loci, I'd appreciate it! From higginsb78 from gmail.com Wed Jan 28 10:37:11 2009 From: higginsb78 from gmail.com (Brian P Higgins) Date: Wed Jan 28 12:36:36 2009 Subject: Electroporation In-Reply-To: <77ddf6c70901272215x2f1c9aceo9bfcd3224c08341d@mail.gmail.com> References: <77ddf6c70901272215x2f1c9aceo9bfcd3224c08341d@mail.gmail.com> Message-ID: You could try drop-dialyzing the sample on a 0.22 or 0.45 micron filter first. The filters come as small, 1-inch diameter circles. I partially fill a petri dish with ddh20, then drop the filter in. I then put 3-5ul of the ligation reaction (after deactivation) on the filter and let it sit for 5-10min. After that, I pick up 1ul and transform via electroporation. This should work. Some ligase mixes (like NEB's QuickLigase) contain Ligases that can't be heat inactivated, so a column or precipitation would be better. In addition, some include PEG and other constituents that can't be removed easily, but will hinder the transformation if not taken out. You can try a column spin as well, but the recovery is probably comparable to a precipitation. I sometimes use the Zymo clean and concentrator or the Qiagen Minelute kits b/c they have very small elution volumes (<10ul). On Wed, Jan 28, 2009 at 1:15 AM, yoginee budhkar wrote: > Dear All, > > I want to use biorad gene pulser for transforming *E. coli* with my ligated > mix. > > It is mentioned in the bio rad manual that we must incubate the ligated mix > at 65deg C for 15 min to inactivate the enzyme and then proceed with its > dilution or etOH precipitation to get rid of extra salts that may decrease > the resistance and cause arcing. > > I did go for the boiling step, but I'm afraid I'll lose the DNA by > precipitation since already only about 80 to 100 ng, insert + vector DNA is > present in my ligated mix. Cannot afford to lose any of it during > precipitation > > What could be done to get rid of the salt content that hampers the > electroporation? > > -- > -- > Yoginee Budhkar > Junior Research Fellow > Food Engineering and Technology Department > Institute of Chemical Technology > Matunga, Mumbai 400019 > Contact: +91 9223355677 > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From depbioakbari from yahoo.com Wed Jan 28 03:59:22 2009 From: depbioakbari from yahoo.com (neda akbari) Date: Wed Jan 28 12:37:02 2009 Subject: Isolation of cell membrane protein Message-ID: <986296.15158.qm@web50211.mail.re2.yahoo.com> i want to isolate an enzyme which bound to cell membrane. i used Tris-buffer with triton X-100 ( 0.5 %) pH= 8 by sonication but most of them didnt isolated by comparing with SDS-PAGE.can you help me? thank you in advance From nobody from nospam.not Wed Jan 28 12:55:13 2009 From: nobody from nospam.not (Han) Date: Wed Jan 28 15:03:42 2009 Subject: Isolation of cell membrane protein References: Message-ID: neda akbari wrote in news:mailman.1614.1233164292.29717.methods@net.bio.net: > i want to isolate an enzyme which bound to cell membrane. i used > Tris-buffer with triton X-100 ( 0.5 %) pH= 8 by sonication but most of > them didnt isolated by comparing with SDS-PAGE.can you help me? thank > you in advance > It's possible the enzyme isn't soluble in Triton, or that it requires more than 0.5%. Many membrane proteins are insoluble in Triton. One of the ways to prepare cytoskeletal proteins is to remove the Titon-soluble proteins, and then study what's left. -- Best regards Han email address is invalid From arollie from chla.usc.edu Wed Jan 28 14:42:14 2009 From: arollie from chla.usc.edu (Rollie, Adrienne) Date: Wed Jan 28 15:53:06 2009 Subject: salmon sperm DNA and glycogen Message-ID: <3A68F9182712584B9225E2D0022A477003343167@CHLAEXVS02.LA.AD.CHLA.ORG> Hello, I am wondering about the interchangeability of salmon sperm DNA and glycogen as 'carriers'. I thought that these play different roles, but have recently been told that they can both be used for certain applications. I will be eluting DNA off of a streptavidin bead/biotinylated probe for PCR, and the elution buffer protocol calls for salmon sperm DNA. I'm curious if this would be one of the times, but since I'm not precipitating the DNA and the 'carrier' function here is more likely to override any non-specific nucleotide binding to the probes or beads, I'm not sure. Any info would be appreciated. Thanks! ------------------------------------------------------------------------------ CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential or legally privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of this original message. ============================================================================== From blackhole from abuse.plus.com Thu Jan 29 04:18:10 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Jan 29 11:50:53 2009 Subject: Low concentration miniprep References: <6Slj9PC8uEdJFABp@abuse.plus.com> <6u1gnhFcaahfU2@mid.individual.net> Message-ID: <0hDQRKBSRXgJFA6Z@abuse.plus.com> Historians believe that in newspost on Wed, 28 Jan 2009, DK penned the following literary masterpiece: >Methicillin is not made by any one anymore. I doubt anyone sells it. >Was it in any way better than carbenicilin? It worked as well and was a fraction of the price. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From prae from gmx.net Thu Jan 29 04:25:46 2009 From: prae from gmx.net (Christian Praetorius) Date: Thu Jan 29 11:51:04 2009 Subject: Electroporation References: Message-ID: <6udb0pFeoar3U1@mid.individual.net> dk@no.email.thankstospam.net (DK) wrote: >The best and easiest thing is to do nothing. 1 ul of ligation mix >is perfectly OK with normal electroporation. Thats my experience, too. Making too much effort doesn't make sense. Christian -- X-no-Sig: yes From engelbert_buxbaum from hotmail.com Fri Jan 30 08:50:20 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jan 30 11:28:19 2009 Subject: Doubt regd about packing a column References: Message-ID: Am 24.01.2009, 00:02 Uhr, schrieb Arul Narayanasamy : > I have a doubt about packing a AAL(Lectin bed) to pierce > centrifuge column,5ml,product number-89897.Kindly let me know how to pack > the AAL bed in to this column. A good book on lab procedures or specifically on chromatography should explain the process of packing a column. Pharmacia had a nice booklet on gel filtration that goes over the process in minute detail, it is now available as pdf-file from the GE homepage www.gelifesciences.com/protein-purification From engelbert_buxbaum from hotmail.com Fri Jan 30 09:13:05 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Jan 30 11:28:26 2009 Subject: filter for heme References: Message-ID: Am 27.01.2009, 18:59 Uhr, schrieb Jiang, Xiaoxu : > I am a PhD student. I am working on a isotope labeled heme uptake > experiment by bacteria cells. Now I have a problem about the filter for > heme: after I incubate my cells with heme in medium, I am trying to > filter out the unbound heme and take the membrane filter to the gamma > counter with the cells on it. But I find there are always a lot of > unbounded heme sticks to the membrane and generates a high background, > which pretty much ruins the experiments. I have tried several different > kinds of membrane filters and right now I am using celluloseacetat > filter but the background is still quite high. Is the heme bound to surface receptors of the bacteria or inside them? In the latter case you can bring the background down by extensive washing with a buffer containing "cold" heme. If the former, you have to be more carefull, depending on the k_off washing may be possible, but I'd leave out the cold heme. In any case you want a membrane with low binding for heme (try polysufone), and you may want to preincubate the membrane with a heme solution. I suspect however that most of the unbound heme you see is in the fluid space between the cells, so washing is the only solution. Some others have already suggested spinning the bacteria through a gradient. I have done this with erys, using butyl phtalate (e.g. from Fluka) as separation medium. Make a gradient of 120 ul 3 M KOH, 500 ul butyl phtalate and add up to 200 ul sample. Spin 30 sec at maximum speed (16,000 g) in an Eppendorf centrifuge. Remove the sample and most of the butyl phtalate by aspiration (collecting the waste in a Wulff-bottle). You measure the radioactivity in the KOH phase. From yasirphr from yahoo.com Fri Jan 30 03:03:11 2009 From: yasirphr from yahoo.com (muhammad yasir) Date: Fri Jan 30 13:10:07 2009 Subject: Polyamines analysis on TLC from bacteria In-Reply-To: <200901281711.n0SHBW817519@net.bio.net> Message-ID: <281184.29784.qm@web55908.mail.re3.yahoo.com> -