From C.P.Pena-Diaz from 2006.hull.ac.uk Sat Nov 1 06:31:33 2008 From: C.P.Pena-Diaz from 2006.hull.ac.uk (Carmen P Pena Diaz) Date: Sat Nov 1 12:04:34 2008 Subject: Problems using pGEX - other expression vector? References: <5696f7c50810290251m52aa93c8we751d636b59bec67@mail.gmail.com> Message-ID: <445800D8FD5D3D43878FD7D2842A304743540A@EXCL2VS2.adir.hull.ac.uk> But is your problem about expression or about cloning? -----Original Message----- From: methods-bounces@oat.bio.indiana.edu on behalf of Daniela Schwoerer Sent: Wed 29/10/2008 09:51 To: methods@magpie.bio.indiana.edu Subject: Problems using pGEX - other expression vector? Hi everyone, I tried to express a protein using pGEX-4T-3 vector which provides a tag for GST-fusion proteins. Unfortunately it doesn't seem to work and also technical support is not helpful. I tried everything, I believe the multicloningsite ist mutated. Or its the moon's fault, who knows. Does anyone use this vector and did you also have problems when trying to clone a gene? (i used BamHI and XhoI restriction sites) Does anyone have an idea which other vector i could use, most likely also with a GST-tag for fusion proteins? Thanks for the help! _______________________________________________ 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 shaakanin from yahoo.com Sat Nov 1 17:53:35 2008 From: shaakanin from yahoo.com (Jess) Date: Sat Nov 1 19:05:20 2008 Subject: Problems using pGEX In-Reply-To: <200811011706.mA1H6oV23932@net.bio.net> Message-ID: <847571.97779.qm@web52908.mail.re2.yahoo.com> 1. Re: Problems using pGEX Ok this could be long: There are so many different problems you could be having with this expression. Some of these are going to sound like stupid questions, however, I think this is where to start: #1) You said you had problems with cloning, are you sure your gene is present? #2) Have you also sequenced your gene to make sure you don't have nonsense mutations? #3) Are you sure you're in the right reading frame? You say you "don't get" your protein, but does that mean you're getting GST and not your protein, or you're not getting GST either? #4) What growth conditions have you tried? (Media, temperature, induction, additives) Just because the plasmid is there, and you have the right sequence and you're in the right reading frame- doesn't mean that your protein will automatically be there. Make sure 1-3 are answered and let me know what growth conditions you've tried and let's go from there. I am happy to help with any protein problem- and I understand just how frustrating something like this can be. You may also have a toxic protein, or heterologous isseus, which may have to be solved a different way. Sometimes yeast, insect cell, gram positive bacteria any of these can work while e coli does not. Cheers and best wishes Jess ? Grad. Res. Asst. University of Texas- Austin Institute for Cellular and Molecular Biology Department of Medicinal Chemistry ?????????? -Hook 'em Horns! ? From eenigoy from gmail.com Mon Nov 3 06:00:45 2008 From: eenigoy from gmail.com (yoginee budhkar) Date: Mon Nov 3 12:34:52 2008 Subject: Hand held UV torch Message-ID: <77ddf6c70811030300n716085d9r5a693cbc204c4420@mail.gmail.com> Has any one used a handheld uv torch to look at DNA bands in a gel? If yes, could you tell me the manufacturers? --Yg From allison from nospam.com Mon Nov 3 14:58:19 2008 From: allison from nospam.com (Allison) Date: Mon Nov 3 21:18:33 2008 Subject: source for microscope parts? Message-ID: <490f1ec5$0$5543$9a6e19ea@news.newshosting.com> We have a Leitz Diavert microscope that is in good working condition except for the fact that it is missing a phase ring. Unfortunately, the microscope was bought in the 80's and is obsolete as far as Leica is concerned....so the part number for the phase ring is no longer in their system. Does anyone know of a good source to get parts for an old microscope? TIA Allison From allison from nospam.com Tue Nov 4 10:45:04 2008 From: allison from nospam.com (Allison) Date: Tue Nov 4 15:58:07 2008 Subject: source for microscope parts? In-Reply-To: References: <490f1ec5$0$5543$9a6e19ea@news.newshosting.com> Message-ID: <491034e2$0$5518$9a6e19ea@news.newshosting.com> Dr Engelbert Buxbaum wrote: > Am 03.11.2008, 15:58 Uhr, schrieb Allison : > >> We have a Leitz Diavert microscope that is in good working condition >> except for the fact that it is missing a phase ring. Unfortunately, >> the microscope was bought in the 80's and is obsolete as far as Leica >> is concerned....so the part number for the phase ring is no longer in >> their system. Does anyone know of a good source to get parts for an >> old microscope? > > Since you do not say where you are this answer has to be broad: > Microscopic Societies often have ways for selling and buying microscope > parts, like ads in their journals or web pages. Especially for high > value brands like Leitz and Zeiss you can get spare parts this way for > many decades. There are also companies that specialise in used lab > equipment. And of course a simple web-search for the part number may > always turn up something. FWIW, I'm in Canada so I figure a Canadian or American source would be the easiest for me. (I tried a web search for the part number with no luck, probably because the microscope was bought in pre-Web days!) I am inquiring at used lab equipment sites but so far no luck there either. From engelbert_buxbaum from hotmail.com Tue Nov 4 10:31:03 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Nov 4 15:58:13 2008 Subject: Hand held UV torch References: Message-ID: Am 03.11.2008, 07:00 Uhr, schrieb yoginee budhkar : > Has any one used a handheld uv torch to look at DNA bands in a gel? > If yes, could you tell me the manufacturers? Ultraviolet Products Inc (http://www.uvp.com/) is one place where you can go, I had good experiences with their MineralLight handlamp. From engelbert_buxbaum from hotmail.com Tue Nov 4 10:35:32 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Nov 4 15:58:20 2008 Subject: source for microscope parts? References: <490f1ec5$0$5543$9a6e19ea@news.newshosting.com> Message-ID: Am 03.11.2008, 15:58 Uhr, schrieb Allison : > We have a Leitz Diavert microscope that is in good working condition > except for the fact that it is missing a phase ring. Unfortunately, the > microscope was bought in the 80's and is obsolete as far as Leica is > concerned....so the part number for the phase ring is no longer in their > system. Does anyone know of a good source to get parts for an old > microscope? Since you do not say where you are this answer has to be broad: Microscopic Societies often have ways for selling and buying microscope parts, like ads in their journals or web pages. Especially for high value brands like Leitz and Zeiss you can get spare parts this way for many decades. There are also companies that specialise in used lab equipment. And of course a simple web-search for the part number may always turn up something. From bmacgreg from unc.edu Tue Nov 4 14:00:20 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Tue Nov 4 15:58:28 2008 Subject: Handheld UV In-Reply-To: <200811041705.mA4H5WV23730@net.bio.net> References: <200811041705.mA4H5WV23730@net.bio.net> Message-ID: > > > From: "yoginee budhkar" > Date: November 3, 2008 6:00:45 AM EST > To: methods@magpie.bio.indiana.edu > Subject: Hand held UV torch > > > Has any one used a handheld uv torch to look at DNA bands in a gel? > If yes, could you tell me the manufacturers? > --Yg Hi Yg, There are probably at least several, but here's one: http://www.uvp.com/handheldlamps.html . Barbara MacGregor From tracyscience from gmail.com Tue Nov 4 14:51:37 2008 From: tracyscience from gmail.com (TC) Date: Tue Nov 4 15:59:46 2008 Subject: running ssDNA (30-1000nt) in denatured conditions Message-ID: <2387671c0811041151o736c22d0l2d5be578dbb67567@mail.gmail.com> Hi all, I am trying to run DNA under denatured conditions. Has anyone run single stranded DNA (30nt to 1000nt) under denatured conditions. I am trying to decide whether to run a low percentage urea acrylamide gel or an alkaline agarose gel. Any thoughts/ideas? I am thinking maybe running a low percentage (3-4%) urea acrylamide gel, but wonder that it may not be able to resolve ssDNA above couple hundred nucleotides in length even in low acrylamide such as 3-4%? Any thoughts? Has anyone run alkaline agaorse for ssDNA 30nt-1000nt? If so, may I ask for your conditions? THANKS! Tracy From gallaght from umbi.umd.edu Wed Nov 5 10:49:10 2008 From: gallaght from umbi.umd.edu (Theresa Gallagher) Date: Wed Nov 5 13:11:46 2008 Subject: Help with protein precipitation Message-ID: <000001c93f5e$11439fd0$33cadf70$@umd.edu> Hi, I am purifying 6-His tagged proteins from inclusion bodies using Talon resin. All goes well until I try and dialyze out the 8 M urea. I have tried two methods: 1. Step wise dialysis from 8M urea to 0M (in hepes buffer) when the dialysis buffer reached 2M the whole lot precipitates out into the cassette 2. Using decreasing concentrations of urea to wash the column - when it came to elution time I had no protein eluted. If anyone could give me some advice on additives for the urea or a method that would be more suitable I would appreciate it! Thanks in advance! From broxxx from gmail.com Wed Nov 5 17:06:51 2008 From: broxxx from gmail.com (Brock Schuman) Date: Wed Nov 5 17:47:33 2008 Subject: cold room resistant pump Message-ID: <39d1b5aa0811051406k42a5b844u7a31b89c89cd2323@mail.gmail.com> I've been having the same problem about pumps wearing down in the cold room. Our old Gilson fraction collector works great in there, but the GE pumps we have been using have a lot of open circuitry that tends to get moist/salty. Just looking into it now, the mandel/gilson pumps look pretty solid, but I haven't heard any feedback on them. You find any cheap solutions yet? --------------------------------------------------- --------------------------------------------------- Brock Schuman, Graduate Student Department of Biochemistry & Microbiology University of Victoria PO Box 3055 STN CSC Victoria, BC, V8W 3P6 CANADA tel: 250-721-8945 FAX: 250-721-8855 From cjtcdy from gmail.com Tue Nov 11 00:42:24 2008 From: cjtcdy from gmail.com (chiranjit chowdhury) Date: Tue Nov 11 12:29:00 2008 Subject: kind help please Message-ID: <2021705c0811102142y78d32fddj545c4ee5beed7a31@mail.gmail.com> Can anybody please tell me whether bacterial periplasm contains ATP? -- Chiranjit Chowdhury Department of Biotechnology Indian Institute of Technology, Kharagpur Kharagpur, West Bengal, India Pin: 721302 Official email: chiranjit.chowdhury@iitkgp.ac.in From sjvinay from yahoo.com Tue Nov 11 05:28:19 2008 From: sjvinay from yahoo.com (vinay sj) Date: Tue Nov 11 12:29:15 2008 Subject: Gel analysis of ligation product Message-ID: <567318.45690.qm@web54203.mail.re2.yahoo.com> Hello, I have a question about gel analysis of ligation product. I would like to insert a 1.7 Kb fragement into a 3.3 Kb vector. I have done the restrictions. I would like to do a gel analysis before transforming to save me time with mini preps. I have done around 10 ligations so far. On the gel, I see that some times I get blanks(i.e no vector, no insert and no ligated products). Some times I get ladders of various combinations of vectors and inserts. These ladders do not transform and I do not get any colonies. Now, my question is if the blanks are the optimal ligation products ? I read in his forum that since ligated products have varying winding numbers, they are not to be seen on a gel. Can anyone confirm this ? I know that nicked circles migrate slowly on a gel and I was expecting a slowly moving band, which I was planning on purifying and transforming. But so far I have not been able to get this. Any help would be appreciated. Regards, Vinay. From hroychow from nmsu.edu Tue Nov 11 12:42:10 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Tue Nov 11 14:51:40 2008 Subject: Gel analysis of ligation product In-Reply-To: <567318.45690.qm@web54203.mail.re2.yahoo.com> References: <567318.45690.qm@web54203.mail.re2.yahoo.com> Message-ID: <2268.128.123.174.0.1226425330.squirrel@webmail.nmsu.edu> In my opinion, gel analysis of ligated products is a step that wastes time and resources. Transformation is an overnight process, and individual colonies can be checked by either a simple PCR or by a quick and dirty plasmid prep. Ligation products may or may not show up on a gel, depending upon how the gel is prepared and run. Besides, one is simply wasting the ligation products by running a gel. > Hello, > > I have a question about gel analysis of ligation product. I would like to > insert a 1.7 Kb fragement into a 3.3 Kb vector. I have done the > restrictions. I would like to do a gel analysis before transforming to > save me time with mini preps. I have done around 10 ligations so far. On > the gel, I see that some times I get blanks(i.e no vector, no insert and > no ligated products). Some times I get ladders of various combinations of > vectors and inserts. These ladders do not transform and I do not get any > colonies. Now, my question is if the blanks are the optimal ligation > products ? I read in his forum that since ligated products have varying > winding numbers, they are not to be seen on a gel. Can anyone confirm this > ? > > I know that nicked circles migrate slowly on a gel and I was expecting a > slowly moving band, which I was planning on purifying and transforming. > But so far I have not been able to get this. > > Any help would be appreciated. > > Regards, > Vinay. > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From mlsulliv from wisc.edu Tue Nov 11 13:31:08 2008 From: mlsulliv from wisc.edu (Michael Sullivan) Date: Tue Nov 11 14:51:45 2008 Subject: Gel analysis of ligation product In-Reply-To: <567318.45690.qm@web54203.mail.re2.yahoo.com> References: <567318.45690.qm@web54203.mail.re2.yahoo.com> Message-ID: <9BF7032B-6607-4476-9A97-E3C65B6007FB@wisc.edu> Vinay, My experience is that doing anything with a ligation reaction other than transforming it is a waste of time. You might get some slight amount of information from looking at the reaction products compared to what went into the reaction, but my experience is that what you see on the gel doesn't look like anything you'd expect corresponding to your clone of interest. Basically you'll just find out that the ligase changed what the fragments look like on a gel and what is a correct clone may only be a minor product in the reaction. As far as gel purifying and transforming a putative ligation product: I'd suggest not trying that. All you'll do is expose your product to additional UV radiation (and possibly making it non-replicable once you transform) and losing material. My opinion is that your best bet is to transform and find your clone later. All told, minipreps and restriction digests are trivial. If you have trouble recovering a clone, there's probably some fundamental problem with the cloning strategy or ligase reaction. The latter is more easily determined by running a control, not analyzing ligation products. Hope this helps. Mike On Nov 11, 2008, at 4:28 AM, vinay sj wrote: > Hello, > > I have a question about gel analysis of ligation product. I would > like to insert a 1.7 Kb fragement into a 3.3 Kb vector. I have done > the restrictions. I would like to do a gel analysis before > transforming to save me time with mini preps. I have done around 10 > ligations so far. On the gel, I see that some times I get blanks > (i.e no vector, no insert and no ligated products). Some times I > get ladders of various combinations of vectors and inserts. These > ladders do not transform and I do not get any colonies. Now, my > question is if the blanks are the optimal ligation products ? I > read in his forum that since ligated products have varying winding > numbers, they are not to be seen on a gel. Can anyone confirm this ? > > I know that nicked circles migrate slowly on a gel and I was > expecting a slowly moving band, which I was planning on purifying > and transforming. But so far I have not been able to get this. > > Any help would be appreciated. > > Regards, > Vinay. > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From straube from biochem.mpg.de Thu Nov 13 09:06:58 2008 From: straube from biochem.mpg.de (Werner Straube) Date: Thu Nov 13 11:06:28 2008 Subject: Volatile Buffers covering pH 3 - 10 Message-ID: <36B75E885C4376419EC0020DA151B41CE6CA33@msx.w2k.biochem.mpg.de> Hello everybody, I am looking for a list/overview of volatile buffers (and mass spec-compatible) which cover the range from pH 3 - 10. Does anyone can help me or know a good reference ? Thank you, Werner ********************************************* Werner L. Straube From nick.theodorakis from gmail.com Fri Nov 14 08:23:03 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri Nov 14 11:55:20 2008 Subject: Volatile Buffers covering pH 3 - 10 References: Message-ID: On Nov 13, 8:28 pm, d...@no.email.thankstospam.net (DK) wrote: > In article , "Werner Straube" wrote: > > >I am looking for a list/overview of volatile buffers (and mass > >spec-compatible) which cover the range from pH 3 - 10. Does anyone can > >help me or know a good reference ? > > Formate, acetate, carbonate as buffers and pyridine and > ammonium as counterions will cover your entire range. > > No idea about mass-spec compatibility. > > DK Pyridine has a terrible nose compatibility, though. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From engelbert_buxbaum from hotmail.com Fri Nov 14 10:59:21 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Fri Nov 14 11:55:32 2008 Subject: Volatile Buffers covering pH 3 - 10 References: Message-ID: Am 14.11.2008, 09:23 Uhr, schrieb Nick Theodorakis : >> Formate, acetate, carbonate as buffers and pyridine and >> ammonium as counterions will cover your entire range. > Pyridine has a terrible nose compatibility, though. I have used triethanolammonium bicarbonate for ion exchange and and ion pairing chromatography for the preparation of nucleotide analogues. That's only slightly fishy (contary to triethylamine). From biocjh from gmail.com Fri Nov 14 12:45:09 2008 From: biocjh from gmail.com (jh) Date: Fri Nov 14 15:05:20 2008 Subject: Help with RNAi on virus gene Message-ID: Dear all, I am intended to do RNAi and I write to seek for help that what I should to employ for positive control? What I want to silence is a virus gene. Any suggestions would be appreciated. best regards! Jianhao, Cao From stxsz1 from nottingham.ac.uk Fri Nov 14 19:50:48 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Sat Nov 15 12:40:27 2008 Subject: what protein-protein interaction can lead to irreversible/covalent binding References: <200811141704.mAEH44V01961@net.bio.net> Message-ID: Hi all I want to find a method to pulldown a target protein A indirectly. (it just has to be indirect) I am thinking: to express protein A with epitope tag x first (A-x), then express a second protein B with another epitope tag y (B-y). Protein B will interact with tag x and Protein A can then be co-purified using the epitope tag y, which is fused to protein B. For tag x and protein B, I only got 2 pairs of candidates: IgG and protein A/G; N- and C-terminal of the fluorescent protein. Or use the biotin ligase as protein B and fuse the recognition sequence to protein A as tag x. The ideal tag x and protein B also have to be small so they wont interfere with the function of protein A. Their interaction has to be strong as well. I worked on plants and I know our techniques are always a bit behind animal sciences. So you guys must have a better systems to do this. But please keep in mind that this method has to be two-step and indirect! Thanks in advance SZ 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 stxsz1 from nottingham.ac.uk Sat Nov 15 06:26:36 2008 From: stxsz1 from nottingham.ac.uk (Zhong Silin) Date: Sat Nov 15 12:40:44 2008 Subject: what is the strongest protein-protein interaction? Message-ID: Hi all I want to find a method to pulldown a target protein A indirectly. (it just has to be indirect) I am thinking: to express protein A with epitope tag x first (A-x), then express a second protein B with another epitope tag y (B-y). Protein B will interact with tag x and Protein A can then be co-purified using the epitope tag y, which is fused to protein B. For tag x and protein B, I only got 2 pairs of candidates: IgG and protein A/G; N- and C-terminal of the fluorescent protein. Or use the biotin ligase as protein B and fuse the recognition sequence to protein A as tag x. The ideal tag x and protein B also have to be small so they wont interfere with the function of protein A. Their interaction has to be strong as well. I worked on plants and I know our techniques are always a bit behind animal sciences. So you guys must have a better systems to do this. But please keep in mind that this method has to be two-step and indirect! Thanks in advance SZ 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 biocjh from gmail.com Sun Nov 16 07:36:15 2008 From: biocjh from gmail.com (jh) Date: Sun Nov 16 13:43:23 2008 Subject: Help with RNAi on virus gene In-Reply-To: References: Message-ID: Thank you, would you like to recommend some reference papers on that? 2008/11/15 Giovanni Parisi : > the best positive control I use is realtime PCR or immunoblot > On 14/nov/08, at 18:45, jh wrote: > >> Dear all, >> >> I am intended to do RNAi and I write to seek for help that what I >> should to employ for positive control? >> What I want to silence is a virus gene. Any suggestions would be >> appreciated. >> >> best regards! >> >> Jianhao, Cao >> >> _______________________________________________ >> Methods mailing list >> Methods@net.bio.net >> http://www.bio.net/biomail/listinfo/methods >> > > From shifalich from rediffmail.com Sun Nov 16 22:48:19 2008 From: shifalich from rediffmail.com (shifali chatrath) Date: Mon Nov 17 11:53:23 2008 Subject: what is the strongest protein-protein interaction? Message-ID: <20081117034819.5335.qmail@f4mail-235-140.rediffmail.com> ? HI! Avidin-Biotin interation is strongest known interaction till date. It has affinity constant of femtomolar range, followed by antigen antibody interaction which is of picomolar range on an average. You can try tagging protein A with Biotin and B with Avidin.It wont interfere with protein function if yours are plant proteins. Biotin is Vit.B2. hope it helps. Regards Shifali On Sat, 15 Nov 2008 Zhong Silin wrote : >Hi all > >I want to find a method to pulldown a target protein A indirectly. (it just has to be indirect) > >I am thinking: to express protein A with epitope tag x first (A-x), then express a second protein B with another epitope tag y (B-y). Protein B will interact with tag x and Protein A can then be co-purified using the epitope tag y, which is fused to protein B. > >For tag x and protein B, I only got 2 pairs of candidates: IgG and protein A/G; N- and C-terminal of the fluorescent protein. Or use the biotin ligase as protein B and fuse the recognition sequence to protein A as tag x. > >The ideal tag x and protein B also have to be small so they wont interfere with the function of protein A. Their interaction has to be strong as well. > >I worked on plants and I know our techniques are always a bit behind animal sciences. So you guys must have a better systems to do this. But please keep in mind that this method has to be two-step and indirect! > >Thanks in advance >SZ > >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. > >_______________________________________________ >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 nick.theodorakis from gmail.com Tue Nov 18 13:44:48 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Tue Nov 18 13:47:09 2008 Subject: what is the strongest protein-protein interaction? References: Message-ID: On Nov 16, 10:48?pm, "shifali chatrath" wrote: > ?? > HI! > Avidin-Biotin interation is strongest known interaction till date. It has affinity constant of femtomolar range, followed by antigen antibody interaction which is of picomolar range on an average. > You can try tagging protein A with Biotin and B with Avidin.It wont interfere with protein function if yours are plant proteins. Biotin is Vit.B2. > hope it helps. > Biotin is not a protein, and the OP was looking for an epitope tag to put on A. I don't know of an easy way to biotinylate one specific protein during biosynthesis. Also, vitamin B2 is riboflavin, not biotin. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From cutest_chemist from yahoo.com Wed Nov 19 03:10:16 2008 From: cutest_chemist from yahoo.com (cutest_chemist@yahoo.com) Date: Wed Nov 19 12:59:10 2008 Subject: running ssDNA (30-1000nt) in denatured conditions References: Message-ID: On Nov 5, 12:51?am, TC wrote: > Hi all, > > I am trying to run DNA under denatured conditions. Has anyone run single > stranded DNA (30nt to 1000nt) under denatured conditions. > > I am trying to decide whether to run a low percentage urea acrylamide gel or > an alkaline agarose gel. ?Any thoughts/ideas? ?I am thinking maybe running a > low percentage (3-4%) urea acrylamide gel, but wonder that it may not be > able to resolve ssDNA above couple hundred nucleotides in length even in low > acrylamide such as 3-4%? ?Any thoughts? ?Has anyone run alkaline agaorse for > ssDNA 30nt-1000nt? ?If so, may I ask for your conditions? > > THANKS! > > Tracy Hi... I m currently using 20% Polyacrylamide gel(PAGE)for ssDNA (20-30 nt) and 15% PAGE for ssDNA of 30-50mer.Its giving good results...Hope this will help u..For more lengthy strans i think u should try 8-10% PAGE. Regards From wfeng from u.washington.edu Wed Nov 19 01:50:17 2008 From: wfeng from u.washington.edu (Wenyi Feng) Date: Wed Nov 19 13:34:15 2008 Subject: In-gel end repair and linker ligation Message-ID: Hello, Does anyone have experience with performing end-repair and adaptmer ligation of chromosomal DNA embedded in agarose plugs? I would appreciate any suggestions for products as well as protocols. Thank you in advance. Wenyi Feng From straube from biochem.mpg.de Wed Nov 19 13:52:03 2008 From: straube from biochem.mpg.de (Werner Straube) Date: Wed Nov 19 18:47:34 2008 Subject: Affinity matrix for Arginine Message-ID: <36B75E885C4376419EC0020DA151B41CE6CC00@msx.w2k.biochem.mpg.de> Hello everybody, I am looking for an affinity matrix that binds with high specificity to a C-terminal Arginine, but the binding should be also reversible. Any suggestions ? Thank you, Werner ********************************************* Werner L. Straube From ebs15242 from creighton.edu Wed Nov 19 16:54:10 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Wed Nov 19 18:47:43 2008 Subject: alkaline lysis -- unrealistic spec readings Message-ID: <49248B02.7020207@creighton.edu> We ran out of miniprep columns, so I'm trying out the alkaline lysis protocol in Maniatis (CSH). When checking the DNA on my spec, I get a nice shaped curve, with good ratios, but the concentration is unrealisticly high. I'm getting readings of around 2 ug/ul. When I run it on a gel and actually visualize the DNA, it can't be more than 200ng/ul tops. I can't figure this out. My spec still reads known DNA concentrations correctly. There aren't any smears or anything that would indicate genomic DNA or RNA contamination. This DNA digests just fine with EcoRI too. Anyone have any idea what's going on here? I need to have these plasmids sequenced, but I'm not sure what to tell them the concentration is. Thanks -Ed From straube from biochem.mpg.de Thu Nov 20 05:23:29 2008 From: straube from biochem.mpg.de (Werner Straube) Date: Thu Nov 20 13:08:02 2008 Subject: Affinity matrix for Arginine Message-ID: <36B75E885C4376419EC0020DA151B41CE6CC15@msx.w2k.biochem.mpg.de> Hello everybody, I am looking for an affinity matrix that binds with high specificity to a C-terminal Arginine, but the binding should be also reversible. Any suggestions ? Thank you, Werner ********************************************* Werner L. Straube From hroychow from nmsu.edu Thu Nov 20 10:23:43 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Thu Nov 20 13:08:41 2008 Subject: alkaline lysis -- unrealistic spec readings In-Reply-To: <49248B02.7020207@creighton.edu> References: <49248B02.7020207@creighton.edu> Message-ID: <2828.128.123.174.0.1227194623.squirrel@webmail.nmsu.edu> It has been my experience that alkaline lyses do give high yield and conc. The yield is usually better than that from a column. I am not sure why you are not seeing the conentration on a gel. > We ran out of miniprep columns, so I'm trying out the alkaline lysis > protocol in Maniatis (CSH). When checking the DNA on my spec, I get > a nice shaped curve, with good ratios, but the concentration is > unrealisticly high. I'm getting readings of around 2 ug/ul. When I run > it on a gel and actually visualize the DNA, it can't be more than > 200ng/ul tops. > > I can't figure this out. My spec still reads known DNA concentrations > correctly. There aren't any smears or anything that would indicate genomic > DNA or RNA contamination. This DNA digests just fine with EcoRI too. > > Anyone have any idea what's going on here? I need to have these plasmids > sequenced, but I'm not sure what to tell them the concentration is. > Thanks > -Ed > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Hiranya S. Roychowdhury, Ph.D. Asst. Professor, Health & Public Services Dona Ana Community College New Mexico State University Las Cruces, NM 88003 From J-R.Kraehling from tu-braunschweig.de Thu Nov 20 04:24:56 2008 From: J-R.Kraehling from tu-braunschweig.de (=?iso-8859-1?Q?Jan_R._Kr=E4hling?=) Date: Thu Nov 20 13:10:08 2008 Subject: StrepTag-II Antibody Message-ID: <7C406FEC56864A5EA7BF8FCEC944F7C8@Jan> Hi everyone! I want to buy an antibody against StrepTag-II, but there are a lot of supplier, so I don't know which I should test and buy first. Which antibody after your experience is the best? I found the following supplier: - IBA GmbH - Novagen - Novus Biologicals - Raybiotech - ABR-Affinity Bioreagents - Thermo Scientific - Acris Antibodies GmbH - GenScript Corporation - GenWay Biotech - AbD Serotec Thanks for help! Jan From mcrepeau from ucdavis.edu Thu Nov 20 21:06:48 2008 From: mcrepeau from ucdavis.edu (Marc Crepeau) Date: Thu Nov 20 22:44:01 2008 Subject: amp/labelling with Klenow Message-ID: I'm looking at a NimbleGen protocol for DNA amplification/labelling and I'm a little confused about how it works. They start with 1 ug of denatured DNA and add Cy-3- or Cy-5-labelled random 9mers, dNTPs and Klenow fragment. Then they incubate at 37C for two hours. The result is several micrograms of labelled DNA. My question is how do you get greater than two-fold amplification from this isothermal reaction? I figure there must be some strand displacement going on, and I know Klenow has strand displacement activity, but doesn't the displacement require a nick just downstream of the primer binding site? From virashkgupta from gmail.com Fri Nov 21 00:11:22 2008 From: virashkgupta from gmail.com (Virash Gupta) Date: Fri Nov 21 09:06:16 2008 Subject: Methods Digest, Vol 42, Issue 13, alkaline lysis -- unrealistic spec readings Message-ID: Hi, If there is no genomic DNA or RNA contamination, the high spec reading may be due to high protein contamination. Check for A260/A280 ratio. Minipreps are known for high protein contamination- not due tp protocol but due to error in handling. After adding neutralizing buffer- sod/ pot acetate, due not vortex or shake high, just mix by inverting many times, keep in ice for 5 min and spin at 12-13 K for 10 min or in cold centrifuge for 5 min.- here we need a firm pellet. While transferring supernatant, take care in transferring any ppt (full of proteins and genomic DNA). Otherwise your plasmid DNA yield is quite good- so reagents are OK. We all know that it is proper handling of technique which matters in this science. Besides 2 ug/ul DNA (spec reading) is 10 times the 200 ng/ul- it may be just a calculation error- check it by applying appropriate dilution factor. Hope this will solve. All the best. On 11/20/08, 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: running ssDNA (30-1000nt) in denatured conditions > (cutest_chemist@yahoo.com) > 2. In-gel end repair and linker ligation (Wenyi Feng) > 3. Affinity matrix for Arginine (Werner Straube) > 4. alkaline lysis -- unrealistic spec readings (Ed Siefker) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 19 Nov 2008 00:10:16 -0800 (PST) > From: "cutest_chemist@yahoo.com" > Subject: Re: running ssDNA (30-1000nt) in denatured conditions > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > On Nov 5, 12:51 am, TC wrote: > > Hi all, > > > > I am trying to run DNA under denatured conditions. Has anyone run single > > stranded DNA (30nt to 1000nt) under denatured conditions. > > > > I am trying to decide whether to run a low percentage urea acrylamide gel > or > > an alkaline agarose gel. Any thoughts/ideas? I am thinking maybe > running a > > low percentage (3-4%) urea acrylamide gel, but wonder that it may not be > > able to resolve ssDNA above couple hundred nucleotides in length even in > low > > acrylamide such as 3-4%? Any thoughts? Has anyone run alkaline agaorse > for > > ssDNA 30nt-1000nt? If so, may I ask for your conditions? > > > > THANKS! > > > > Tracy > > Hi... > I m currently using 20% Polyacrylamide gel(PAGE)for ssDNA (20-30 nt) > and 15% PAGE for ssDNA of 30-50mer.Its giving good results...Hope this > will help u..For more lengthy strans i think u should try 8-10% PAGE. > > Regards > > > ------------------------------ > > Message: 2 > Date: Tue, 18 Nov 2008 22:50:17 -0800 > From: Wenyi Feng > Subject: In-gel end repair and linker ligation > To: methods@magpie.bio.indiana.edu > Message-ID: > Content-Type: text/plain; charset=US-ASCII; format=flowed; delsp=yes > > Hello, > > Does anyone have experience with performing end-repair and adaptmer > ligation of chromosomal DNA embedded in agarose plugs? I would > appreciate any suggestions for products as well as protocols. Thank > you in advance. > > Wenyi Feng > > > > ------------------------------ > > Message: 3 > Date: Wed, 19 Nov 2008 19:52:03 +0100 > From: "Werner Straube" > Subject: Affinity matrix for Arginine > To: > Message-ID: > <36B75E885C4376419EC0020DA151B41CE6CC00@msx.w2k.biochem.mpg.de> > Content-Type: text/plain; charset="us-ascii" > > Hello everybody, > > > > I am looking for an affinity matrix that binds with high specificity to > a C-terminal Arginine, but the binding should be also reversible. > > > > Any suggestions ? > > > > Thank you, > > > > Werner > > > > ********************************************* > > Werner L. Straube > > > > > > ------------------------------ > > Message: 4 > Date: Wed, 19 Nov 2008 15:54:10 -0600 > From: Ed Siefker > Subject: alkaline lysis -- unrealistic spec readings > To: methods@magpie.bio.indiana.edu > Message-ID: <49248B02.7020207@creighton.edu> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > We ran out of miniprep columns, so I'm trying out the alkaline lysis > protocol in Maniatis (CSH). When checking the DNA on my spec, I get > a nice shaped curve, with good ratios, but the concentration is > unrealisticly high. I'm getting readings of around 2 ug/ul. When I run > it on a gel and actually visualize the DNA, it can't be more than > 200ng/ul tops. > > I can't figure this out. My spec still reads known DNA concentrations > correctly. There aren't any smears or anything that would indicate genomic > DNA or RNA contamination. This DNA digests just fine with EcoRI too. > > Anyone have any idea what's going on here? I need to have these plasmids > sequenced, but I'm not sure what to tell them the concentration is. Thanks > -Ed > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 42, Issue 13 > *************************************** > -- 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 wmisaki from med.mak.ac.ug Fri Nov 21 07:45:41 2008 From: wmisaki from med.mak.ac.ug (wmisaki@med.mak.ac.ug) Date: Fri Nov 21 09:06:52 2008 Subject: Extracting DNA from Bones Message-ID: <20081121124541.5B0927E7B4@mail.mak.ac.ug> Dear Scarr, I read your memo online about a problem you faced time back on extracting DNA from bones. I have a similar case(30 years old) except that they were not in water as your case. Similarly, there are potential relatives(son) that are willing to offer a reference sample. I looked over the internet for a facility that could offer such specialised services but could not find any. I am wondering, could your memo have yielded useful results? Can you help me with this ? Misaki, Wayengera PhD Division of Human and Molecular Genetics Department of Pathology-Makerere University P o Box 7072Kampala, Uganda. TeL: +256782450610 Email: wmisaki@med.mak.ac.ug From editor from gene-quantification.info Fri Nov 21 04:15:14 2008 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Fri Nov 21 09:19:25 2008 Subject: Call for Papers - qPCR 2009 Event Message-ID: Call for Papers - qPCR 2009 Event 4th international qPCR Symposium & Industrial Exhibition & Application Workshop 9 - 13th March 2009, in Freising-Weihenstephan, Technical University of Munich, Weihenstephan, Germany http://talk-and-poster-call.qpcr2009.net/ ---------------------------------------------------------------------------= -- Dear colleagues, dear researchers, dear company representatives, On behalf of the Organisation Committee and the Scientific Board it is a great pleasure to invite you to the 4th International qPCR Symposium & Industrial Exhibition & Application Workshop to be held at the Center of Life Science in Freising Weihenstephan, Technische Universit=E4t M=FCnchen (Germany). The great international interest in the previous meetings qPCR 2004 qPCR 2005 and qPCR 2007 ] with up to 600 participants coming from over 40 countries, and 30 international companies in the qPCR Industrial Exhibition led us to the decision to repeat the Symposium in spring 2009. We have set the date for the qPCR 2009 Event to 9 - 13th March 2009. The event location is the central lecture hall complex and the foyer at TUM (Technical University of Munich) in Freising Weihenstephan, Germany. The TUM and the Biotech region around Munich is part of the largest Biotech cluster in Europe, located close to the Munich airport in the heart of Bavaria. Leading academic researchers and industrial contributors in the field will be participate in the symposium, which will be an arena for fruitful discussions between researchers of different backgrounds. The Symposium Talks, various Poster Sessions, Industrial Exhibition and associated qPCR Application Workshops offer an overview of the present knowledge and future developments in qPCR technology and its wide applications. The symposium will focus on approximately 40-50 lectures presented by internationally recognised experts in their field. The emphasis will be on unbiased, didactic information exchange. Internationally renown speakers will be participating in a lively and exciting programme enabling the valuable exchange of information in the qPCR field. One third of the talks will be presented by selected invited speakers, one third will be selected from the submitted abstracts and one third will be presented by qPCR company R&D representatives. All scientific contributions will be published in the qPCR 2009 Symposium Proceedings. It is a pleasure to announce the Nobel Prize Laureate Kary Mullis in an own session =9325th Anniversary of PCR=94 The qPCR 2009 Event contains several parts: 1. qPCR Symposium taking place March 9 - 11, 2009 =3D> Talk and Poster sessions http://sessions.qpcr2009.net/ =3D> confirmed Speakers http://speakers.qpcr2009.net/ =3D> download FLYER http://www.bioeps.com/qpcr2009/qPCR-200= 9-3rd-announcement.pdf 2. A parallel qPCR Industrial Exhibition taking place March 9 - 11, 2009 3. Followed by four qPCR application Workshops taking place March 12 -13, powered by the TATAA Biocenter Germany - Basis Module qPCR Application Workshop (2-days) - qPCR Biostatistics & Expression Profiling (2-days) - Sample Preparation (2-days) - High Resoultion Melt (day 1) & Immuno-qPCR (day 2) http://workshops.qpcr2009.net/ ---------------------------------------------------------------------------= -- CALL for TALK & POSTERS Deadline =3D> 31st December 2008 Referee process will be finished until 15th January 2009 http://talk-and-poster-call.qpcr2009.net/ Please register and submit your abstract here =3D> http://registration.qpc= r2009.net/ qPCR 2009 Talk & Poster sessions ---------------------------------------------------------------------------= -- Main topic: Diagnostics & Molecular Markers Markers in diagnostic, prognostic, and therapeutic, markers on DNA, RNA, microRNA, protein, and metabolite level, disease markers, tissue specific markers, cancer markers, stem-cells markers, differentiation markers, methylation markers, diagnostic quantification methods, epigenetics, SNP analysis, HRM =3D high resolution melt applications, =85.. Main topic: Diagnostics & Molecular Markers in agricultural and veterinary Science Diagnostics & Molecular Markers in White-, Green-, Blue-, and Brown- Biotechnology and in agricultural and veterinary Science. Marker genes on diagnostic, prognostic, and therapeutic markers on on DNA, RNA, microRNA, pProtein, and metabolite level, in animals and plants, =85 =85 "25th Anniversary of PCR=94 Session held by Nobel Prize Laureate Kary Mullis sponsored by Bioserch Technologies Single-Cell qPCR single-cell sampling, pre-amplification techniques, laser micro dissection, sub-cellular PCR, micro-manipulation of cell clusters, cellular micro injection, FACS spotting, single cell handling, =85=85 RNAi - microRNA - siRNA Applications RNAi mechanism, microRNA extraction, qRT-PCR technologies to detect microRNA, siRNA applications in combination with qRT-PCR, microRNA targets and microRNA precursors, new siRNA manipulation and microRNA technologies, ..... High Throughput quantitative PCR 384 well applications, new high throughput platforms, qPCR robotics, digital PCR, SNP application, gene expression real-time RT-PCR arrays (mRNA and microRNA), quantitative multiplexing, =85.. Pre-analytical Steps Pre-amplification, sampling technologies, DNA / RNA purification, extraction efficiency, DNA / mRNA / microRNA quality control, Reverse Transcription, RT quality control, external references, =85.. Immuno qPCR development, establishment, optimization of immuno-qPCR, innovative immuno qPCR applications, =85.. qPCR NOS Session - Normalization & Optimization & Standardization sponsored by LONZA new types of normalization, one vs. multiple reference genes, genomic DNA as standard, external standards, optimization of the real-time PCR, inhibition of negative effects, optimization of real-time PCR efficiency, qPCR robotics, multiplexing, establishment of DNA / RNA standards, inter-run standards, national and international studies on qPCR standardization, new quantification strategies, ........ qPCR BioStatistics & BioInformatics software applications, data mining, calculation of relative expression, primer and probe design on mRNA and microRNA level, real- time PCR efficiency determination, mathematical modelling, Multivariate expression profiling raw data analysis, statistics in real-time PCR, data management, multiway expression profiling, multiple regression analysis, 3D data visualization, ........ ---------------------------------------------------------------------------= -- Internet based REGISTRATION & ABSTRACT submission platform =3D> http://registration.qpcr2009.net/ ---------------------------------------------------------------------------= -- qPCR 2009 Industrial Exhibition An industrial exhibition will be held during the qPCR 2009 Event from Monday 9th - Wednesday 11th March 2009 in the foyer of the central lecture hall complex (green frame) and in two side rooms S1 and S2 (blue frame). According to the booth location we have 30 booth in different prize categories. They are all grouped around the central lecture hall H14 that can fit up to 650 people. http://exhibition.qpcr2009.net/ From Nikola.Wenta from nottingham.ac.uk Fri Nov 21 09:58:37 2008 From: Nikola.Wenta from nottingham.ac.uk (Nikola Wenta) Date: Fri Nov 21 12:37:54 2008 Subject: StrepTag-II Antibody In-Reply-To: <200811211420.mALEKEV11518@net.bio.net> References: <200811211420.mALEKEV11518@net.bio.net> Message-ID: Hello Jan! IBA GmbH has engineered the Strep tag. To my knowledge, they also provide the best antibody against it: StrepMAB-Classic. Additionally to the specifications in their data sheet, it can also be used for western blots; we can hardly see any background. Have a look here: http://www.iba-go.de/prottools/index.php?prot_p_poly.html . Niko 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 info from noster-it.com Mon Nov 24 02:13:46 2008 From: info from noster-it.com (Dokorek) Date: Mon Nov 24 11:32:53 2008 Subject: Protein Fingerprinting Made Easy Message-ID: <25872d63-3e83-4d9a-a439-647d516a6cee@l39g2000yqn.googlegroups.com> Combining some traditional experimental methods of molecular biology with computational methods of artificial intelligence, a group of researchers from Ruder Bo=9Akovic Instititute and Faculty of Natural Sciences and Mathematics from Zagreb, Croatia, demonstrated a novel approach for producing =91protein fingerprints=92 of diverse tissues. This result could lead to the development of new convenient methods in medical diagnostics. Dokorek Portal to share biological information-data between people http://biospace.ethz.ch From cjtcdy from gmail.com Mon Nov 24 11:22:21 2008 From: cjtcdy from gmail.com (chiranjit chowdhury) Date: Mon Nov 24 11:33:02 2008 Subject: kind help please Message-ID: <2021705c0811240822k1a7524eaw5491c6ba2929c511@mail.gmail.com> Dear all, Can any body tell me how peptidoglycan can be isolated without the help of HPLC? Regards -- Chiranjit Chowdhury Department of Biotechnology Indian Institute of Technology, Kharagpur Kharagpur, West Bengal, India Pin: 721302 Official email: chiranjit.chowdhury@iitkgp.ac.in From gayacharanbio from yahoo.co.in Wed Nov 26 01:04:38 2008 From: gayacharanbio from yahoo.co.in (Gayacharan Prajapati) Date: Wed Nov 26 17:21:14 2008 Subject: (no subject) Message-ID: <146905.78609.qm@web94007.mail.in2.yahoo.com> I want to amplify single strand DNA by a single gene specific primer from genomic DNA. I used thermal cycler for this purpose, but I am unable to get the result. I want suggestions regarding this. Thank you Birendra, P. GAYACHARAN M.Sc. Biotechnology Lab - 210, Rice Stress Molecular Laboratory Centre for Plant Molecular Biology Tamil Nadu Agri. Uinversity, Coimbatore- 641003 INDIA --------------------------------- Add more friends to your messenger and enjoy! Invite them now. From R.Jayakumar from roswellpark.org Wed Nov 26 21:04:36 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Wed Nov 26 23:42:27 2008 Subject: (no subject) References: <146905.78609.qm@web94007.mail.in2.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E23716D0C@VISTA.roswellpark.org> Hi. How is CPMB doing now? I did my MSc there too. Anyway, your question has hardly any information at all to solve your problem. what kind of gene are you trying to amplify and what genomic DNA (bacteria, plant animal etc. etc), primer or primers. what kind of PCR are you trying to do??. Information like that would be very useful to solve your problem. Regards Jay //////////////////////////////\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ Jayakumar R. Nair, Ph.D. Dept. of Immunology Roswell Park Cancer Institute Buffalo, NY 14263 ________________________________ From: methods-bounces@oat.bio.indiana.edu on behalf of Gayacharan Prajapati Sent: Wed 11/26/2008 1:04 AM To: methods@magpie.bio.indiana.edu Subject: (no subject) I want to amplify single strand DNA by a single gene specific primer from genomic DNA. I used thermal cycler for this purpose, but I am unable to get the result. I want suggestions regarding this. Thank you Birendra, P. GAYACHARAN M.Sc. Biotechnology Lab - 210, Rice Stress Molecular Laboratory Centre for Plant Molecular Biology Tamil Nadu Agri. Uinversity, Coimbatore- 641003 INDIA --------------------------------- Add more friends to your messenger and enjoy! Invite them now. _______________________________________________ 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 shifalich from rediffmail.com Wed Nov 26 20:39:36 2008 From: shifalich from rediffmail.com (shifali chatrath) Date: Wed Nov 26 23:42:34 2008 Subject: (no subject) Message-ID: <1227738873.S.3555.24242.f4mail-235-214.rediffmail.com.1227749976.47075@webmail.rediffmail.com> Dear Gayacharan! The amplification that you will be obtaining like this will be geometric and not exponential. That, as may number of cycles, as is the number fold the amplification will be. That means, you cannot use the number of cycles as is used in normal PCR. Also, check the polarity (5' or 3')of ur primer as well as template, if you know primer designing. Also, in order to get amplification of your ss DNA,if you use too many cycles, I doubt the fidelity of the reaction. So, all these factors you need to take into account. All the best for your experiment. Shifali On Thu, 27 Nov 2008 04:04:33 +0530 wrote >I want to amplify single strand DNA by a single gene specific primer from genomic DNA. I used thermal cycler for this purpose, but I am unable to get the result. I want suggestions regarding this. > ? > ?Thank you > ? > ?Birendra, P. > > >GAYACHARAN >M.Sc. Biotechnology >Lab - 210, Rice Stress Molecular Laboratory >Centre for Plant Molecular Biology >Tamil Nadu Agri. Uinversity, >Coimbatore- 641003 >INDIA > ? ? ? >--------------------------------- > Add more friends to your messenger and enjoy! ?Invite them now. >_______________________________________________ >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 cc122 from mole.bio.cam.ac.uk Fri Nov 28 11:33:34 2008 From: cc122 from mole.bio.cam.ac.uk (C Coward) Date: Fri Nov 28 13:32:44 2008 Subject: Efficiency of (electro)transformation in Salmonella Message-ID: Hello all, I'm after opinions/advice on how to get the best transformation efficiency in Salmonella (enteritidis). I'm currently using electroporation but am routinely getting efficiencies around 10^4 cf/ug of DNA. I don't think restriction systems would be reducing my efficiency as the plasmid DNA is prepared from a Salmonella strain (I haven't tested this though, the plasmid DNA is coming from S. typhimurium LB5010). I do need to test the efficiency of re-introducing my plasmid preps into LB5010. I could really do with higher efficiency so any tips? My protcol is as follows 1. Grow a starter O/N in LB@37C 2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5 3. Wash 1X in 20ml ice-cold dH2O 4. Pellet and wash 1X in 1ml ice-cold dH2O 5. Pellet and wash 2X in 1ml ice-cold 10% glycerol 6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at -80 7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse time around 5ms) Cheers! From gnomenm from yahoo.com Sat Nov 29 13:16:18 2008 From: gnomenm from yahoo.com (michael nelson) Date: Sat Nov 29 13:57:54 2008 Subject: protocol for liposome preparation Message-ID: <5649.57732.qm@web111405.mail.gq1.yahoo.com> Hey, guys, I have some difficulties preparing for clear liposomes solutions. Here is what I did, please advise. ?I start off with 0.1g/ml phosphatidylcholine chloroform solution. I then move it into a round bottomed flask and dry it in a rotary evaporator. However, instead of getting thin films on the surface, what I end up with is a large chunk of light yellowish solid. Does that mean I have to use a larger volume of organic solvent? I then dissolve them into buffer and sonicate. Finally, I got some milky suspension. I assume they are multimellar vesicles. My advisor told me that I could sonicate to make them unilamellar and make the solution goes clear. I tried, but didn't work. Could anyone here tell me how to prepare the unilamellar vesicles in a clear solution? More details are better. Thanks! From novalidaddress from nurfuerspam.de Sat Nov 29 15:22:34 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sat Nov 29 17:45:41 2008 Subject: protocol for liposome preparation References: Message-ID: <0f3b6406-03cb-44c1-b7f1-81853b440f99@v4g2000yqa.googlegroups.com> Hi Michael, you might try this: dissolve PC in EtOH and inject into 20 to 50 fold volume of buffer with an insulin syringe (or the narrowest needle you can get) while vortexing. If the EtOH might cause problems downstream, dialyze against buffer. Worked well for PC/Ceramide vesicles for me. best regards, Wo From vasipalli from me.com Sat Nov 29 17:07:59 2008 From: vasipalli from me.com (Mahesh Reddy) Date: Sat Nov 29 17:45:49 2008 Subject: Methods Digest, Vol 42, Issue 18 In-Reply-To: <200811291704.mATH4bV00803@net.bio.net> References: <200811291704.mATH4bV00803@net.bio.net> Message-ID: <6125B7E2-0D8C-4234-BAC3-51468227E452@me.com> I prefer to use XL-10 gold competent cells.you could get from stratagene. Sent from my iPhone On 29 Nov 2008, at 17:04, 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. Efficiency of (electro)transformation in Salmonella (C Coward) > 2. Re: Efficiency of (electro)transformation in Salmonella (DK) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: 28 Nov 2008 16:33:34 GMT > From: C Coward > Subject: Efficiency of (electro)transformation in Salmonella > To: methods@net.bio.net > Message-ID: > > Hello all, > > I'm after opinions/advice on how to get the best transformation > efficiency > in Salmonella (enteritidis). > > I'm currently using electroporation but am routinely getting > efficiencies > around 10^4 cf/ug of DNA. I don't think restriction systems would be > reducing my efficiency as the plasmid DNA is prepared from a > Salmonella > strain (I haven't tested this though, the plasmid DNA is coming from > S. > typhimurium LB5010). I do need to test the efficiency of re- > introducing my > plasmid preps into LB5010. > > I could really do with higher efficiency so any tips? > > My protcol is as follows > > 1. Grow a starter O/N in LB@37C > 2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5 > 3. Wash 1X in 20ml ice-cold dH2O > 4. Pellet and wash 1X in 1ml ice-cold dH2O > 5. Pellet and wash 2X in 1ml ice-cold 10% glycerol > 6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at > -80 > 7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse time > around 5ms) > > Cheers! > > > > ------------------------------ > > Message: 2 > Date: Fri, 28 Nov 2008 17:11:35 GMT > From: dk@no.email.thankstospam.net (DK) > Subject: Re: Efficiency of (electro)transformation in Salmonella > To: methods@net.bio.net > Message-ID: > > In article , C Coward > wrote: >> Hello all, >> >> I'm after opinions/advice on how to get the best transformation >> efficiency >> in Salmonella (enteritidis). >> >> I'm currently using electroporation but am routinely getting >> efficiencies >> around 10^4 cf/ug of DNA. I don't think restriction systems would be >> reducing my efficiency as the plasmid DNA is prepared from a >> Salmonella >> strain (I haven't tested this though, the plasmid DNA is coming >> from S. >> typhimurium LB5010). I do need to test the efficiency of re- >> introducing my >> plasmid preps into LB5010. >> >> I could really do with higher efficiency so any tips? >> >> My protcol is as follows >> >> 1. Grow a starter O/N in LB@37C >> 2. Inoculate 20ml with 100ul of the starter, grow @37C to OD~0.5 >> 3. Wash 1X in 20ml ice-cold dH2O >> 4. Pellet and wash 1X in 1ml ice-cold dH2O >> 5. Pellet and wash 2X in 1ml ice-cold 10% glycerol >> 6. Resuspend in 400ul 10% glycerol, aliquot in 100ul lots, store at >> -80 >> 7. Electroporate using 2mm gap cuvettes, 2kV, 25uF, 200ohm (pulse >> time >> around 5ms) > > 1. Try higher cell concentration. Typical E.coli preps are 1 L > culture --> > resuspend in 2 ml final, i.e. 500X. Yours is only 50X. > 2. Vary voltage. E.g. 1.8, 2.2, 2.4 kV for 2 mm gap, 1.4,1.6,1.8 kV > for 1 mm gap. > 3. Try 7% and 10% DMSO in place of glycerol. > 4. Try growing cultures at 20C instead of 37. Gives a 10X boost > for E.coli. > 5. Try including 50 mM DTT in washes (will require voltage > optimization since when the cell wall is weakened the electric > filed intensity needs to be reduced). > > DK > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 42, Issue 18 > *************************************** From novalidaddress from nurfuerspam.de Sun Nov 30 04:37:58 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sun Nov 30 13:35:28 2008 Subject: Affinity matrix for Arginine References: Message-ID: <417b8963-20be-4360-8905-48f492196efc@v38g2000yqb.googlegroups.com> Hi Werner, depends a bit on the composition of your sample. For cleanup, ion exchange should be a good choice. If you want to isolate proteins with such a motif from a crude lysate, probably an affinity column with an antibody against said motif should be considered. Regards, Wo On Nov 20, 11:23?am, "Werner Straube" wrote: > Hello everybody, > > I am looking for an affinity matrix that binds with high specificity to > a C-terminal Arginine, but the binding should be also reversible. > > Any suggestions ? > > Thank you, > > Werner > > ********************************************* > > Werner L. Straube From novalidaddress from nurfuerspam.de Sun Nov 30 04:49:30 2008 From: novalidaddress from nurfuerspam.de (WS) Date: Sun Nov 30 13:35:52 2008 Subject: Aliquotting NHS-Biotin Message-ID: <9a8539b6-d7c4-4569-a9fa-cca9b00d0081@w35g2000yqm.googlegroups.com> Dear Colleagues, I have a 100mg sample of NHS biotin, currently stored at -20 in a sealed container. I need to distribute and store in several aliquots. I thought of dissolving it in essentially water-free DMSO and store the aliquots again at -20 in small vials equipped with a gasket in the lid. Dos that work resp. what lifetime of the NHS may I expect? Are are there any better alternatives? e.g. dissolving in absolute CH2Cl2 (if soluble) and drying in a speed-vac before storage? Your suggestions are welcome! Wo