From blacknuss from gmail.com Tue Sep 1 03:42:34 2009 From: blacknuss from gmail.com (Joe) Date: Tue Sep 1 12:47:58 2009 Subject: Conditions for use, Tet-On system Message-ID: <4cdf7937-130e-4709-aeb2-c073cd0f9066@h30g2000vbr.googlegroups.com> Back in '97, a student in our lab bought the Tet-On system from Clontech for use in his thesis work. I work in a small biotech company with many research-based activities and with many academic collaborations, including this thesis work. No commercial work was done with the Tet-On system, and the final results were published in 2001. In 2004, the company TET Systems was formed, apparently to profit from the Tet-patents filed by Gossen and Bujard (first patent filed November 1995). Later, TET have contacted us and asked us to enter a license on the system, not only for present and future use, but also past use. As we recall it, there were no licensing conditions attached to our purchase in 1997, but it is difficult for us to dig up that information after 12 years. Does anybody know where I can find a copy of the conditions of use for the Tet-On system dated around spring 1997? Best regards, /JT From aawara from pontiff-playground.org Tue Sep 1 06:11:25 2009 From: aawara from pontiff-playground.org (Aawara Chowdhury) Date: Tue Sep 1 12:48:03 2009 Subject: Conditions for use, Tet-On system References: <4cdf7937-130e-4709-aeb2-c073cd0f9066@h30g2000vbr.googlegroups.com> Message-ID: In <4cdf7937-130e-4709-aeb2-c073cd0f9066@h30g2000vbr.googlegroups.com>, Joe wrote: > Back in '97, a student in our lab bought the Tet-On system from > Clontech for use in his thesis work. I work in a small biotech company > with many research-based activities and with many academic > collaborations, including this thesis work. No commercial work was > done with the Tet-On system, and the final results were published in > 2001. No commercial work may have been performed, but was the Tet-On system purchased from Clontech by a commercial entity, i.e. the small biotech company and not the academic entity the student was affiliated with? If it was the former then TET Systems has obtained that information from Clontech and contacted you on that basis. As far as I remember (from 1999), there were restrictions on commercial use of the Tet-On system. However, last year, I was contacted by TET Systems to complete a new "Notice & Acknowledgement" agreement for non-profit users. It was a little sticky for us, because we have modified the Tet-On system and published manuscripts with the modified system. AC -- Email: echo 36434455860060025978157675027927670979097959886449930P | dc From kuforen from gmail.com Wed Sep 2 21:54:05 2009 From: kuforen from gmail.com (Seong Hwan Park) Date: Wed Sep 2 22:06:23 2009 Subject: Total RNA extraction from whole blood Message-ID: <7e2b93f90909021954n7e49b3f4yb1ec7e1f36ab90a9@mail.gmail.com> Hello. I have to extract total RNA from whole blood using a guanidium-based reagent, RNAiso plus (Takara). Takara says that RNAiso plus is compatible with Trizol. With 1mL of RNAiso plus, RNA from 100ul of whole blood can be processed. In the Takara protocol, expected amount of RNA from 100ul human whole blood is about 1.5~2ug. However, in my case, the yield was quite low(about 150ng) and 28S and 18S rRNA bands disappeared after DNase I treatment. I am stuck in this problem for 1 month. Today I am extracting RNA again with two times of RNAiso plus treatment If this does not work, I have no idea what to do next. Does anybody have an experience with RNA extraction from whole blood using RNAiso plus or Tri Reagent? Thank you. Sincerely, Seong Hwan -- Seong Hwan Park, M.D., Ph.D. Instructor in Legal Medicine, College of Medicine, Korea University 126-1 Anadong 5ga, Seongbukgu, Seoul 136-704 South Korea E-mail: kuforen@gmail.com or pelvis@korea.ac.kr Tel: 82-2-920-6158 Fax: 82-2-928-3901 From jtornoe from gmail.com Thu Sep 3 02:57:48 2009 From: jtornoe from gmail.com (JT) Date: Thu Sep 3 10:04:39 2009 Subject: Conditions for use, Tet-On system References: <4cdf7937-130e-4709-aeb2-c073cd0f9066@h30g2000vbr.googlegroups.com> Message-ID: <01ce8591-aef8-4171-8afa-9973c75fe5cc@z24g2000yqb.googlegroups.com> On Sep 1, 1:11?pm, Aawara Chowdhury wrote: > If it was the former then TET Systems has obtained that information > from Clontech and contacted you on that basis. > > As far as I remember (from 1999), there were restrictions on > commercial use of the Tet-On system. > > However, last year, I was contacted by TET Systems to complete a > new "Notice & Acknowledgement" agreement for non-profit users. It was > a little sticky for us, because we have modified the Tet-On system and > published manuscripts with the modified system. > > AC Thanks AC. The situation is a little sticky for us as well, since we also have modified and published with the system. As we recall it, however, there were no licensing conditions present when we purchased the vectors in Spring 1997 (which was a little more than one year after the first Tet-On patent), that appears to have come later. So, that is why I am very interested in hearing from anybody that would have a copy of the 1997 licensing information / conditions somewhere in their archives. Thanks, /JT From rahimpour_a from yahoo.com Fri Sep 4 02:41:03 2009 From: rahimpour_a from yahoo.com (Azam Rahimpour) Date: Fri Sep 4 11:07:20 2009 Subject: electroporation In-Reply-To: Message-ID: <285506.43257.qm@web52706.mail.re2.yahoo.com> Hi DK I am already looking a way for enhancing electroporation rate on mammalian cells, can you please help me with some protocols or address me to helpfull literature? best --- On Mon, 8/24/09, DK wrote: From: DK Subject: Re: low LPS plasmid prep protocol To: methods@magpie.bio.indiana.edu Date: Monday, August 24, 2009, 8:49 PM In article , aawara@pontiff-playground.org wrote: >In , > Nick Theodorakis wrote: > >> On Aug 23, 4:37?pm, Bari Zahedi wrote: >>> Does anyone have a non-kit plasmid prep protocol for cell transfection that > will generate plasmid with low LPS levels? I will be using the plasmids ?to > transfect immune cells. Thanks! >> >> Doing a double-banding in CsCl after alkaline lysis should be pretty >> low in LPS, but I doubt that's much cheaper than using a commercial >> kit, and you need access to an ultracentrifuge and the appropriate >> rotor. > >This is how my lab does it, using a VTi90 rotor that cost an arm and a leg. >But we get both spins done in one day -? 4 hours spins at 70K. > >After the second spin, it takes us about 6 extractions with saturated >butanol to get the ethidium bromide out.? We precipitate the DNA out of >CsCl using ethanol, rather than an overnight dialysis. > >Typically from a 400 ml culture (in terrific broth), we recover >about 300 - 600 micrograms of pBR322-based low copy plasmids. Wow, so much work for so little plasmid. Just out of curiosity: why low copy plasmids for mammalian transfections? Highly repetitive sequences or what? One would think that simply by swapping an ori you'd get your purity up ~10X. Another thought on LPS removal: LPS has lipid and DNA does not. LPS must bind to resins designed for lipid removal. Bio-Rad, Calbiochem and Pierce sell them. (The polymixin-based sorbents are more selective, way more expensive and are designed to not bind proteins so much and detergent removal ones do; so for DNA purification simple and quite cheap detergent binding resins should do fine). >It is, as you say, a pain.? But on the other hand, we get excellent >transfections of primary cells (lymphocytes in our case) using nucleofactor >electroporations. Nucleofector and nucleofector is a marketing device. There is nothing new or original in it. It's just a simple electroporator, pretty generic HEPES-based buffer and an alkaline peptide that is efficiently targeted to the nucleus. In Amaxa's case, it is most likely MEEDTPPIKKKRKVEDL at 25 microM final, a neutral charge fusion of NLS of SV40 large T-antigen and N-terminal sequence of VP2 protein. The use of DNA-binding peptides (and this one, specifically) to enhance transfection was reported and patented at least 10 and 5 years before the Amaxa's patent, respectively. So Amaxa's patent is just a scam to circumvent existing patents, and the "revolutionary technology" for which absolutely no hint is available throught the company is nothing by a marketing ploy. Not to be an asshole, but I have to ask: Aawara, do you guys always do empty vector transfection controls? In more than one papers I saw "nucleofection" used without such control. How do I know the effects were not due to the magic bufferyou observe after transfections relate to peptide's presense? DK P.S. I have spiked plasmid with histones from Sigma to observe increase of electroporation efficiency 20 years ago; 15 years ago the same was repeated in the lab accross the hall in Paramecium electroporations; neither was ever published, though). _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From cjmcdermottroe from googlemail.com Fri Sep 4 10:22:06 2009 From: cjmcdermottroe from googlemail.com (Chris McDermott-Roe) Date: Fri Sep 4 11:07:25 2009 Subject: protein purification problem Message-ID: Hi all, I'm having problems purifying a myc-tagged protein. My protein of interest is toxic in bacteria so I decided to express it in mammalian cells (HEK293s) and (at least in theory) purify it using an anti-myc antibody agarose resin that I got from Sigma (A 7470) and some SigmaPrep spin columns (MC1000) also from Sigma. Well to cut a long story short, I havent been able to isolate my protein. I dont know if my protein simply isnt sticking to the beads (maybe because the tag is buried in the protein for some reason) or that I can elute it. If anybody has any strategy/reagent suggestions, that would be great. Some details that may be relevant: I've got mounds of protein as I harvested cells and prepared proteins from 5x75 cubic cm flasks so I'm pretty sure it's not an issue of quantity. Second, I my protein does have the tag; I've verified this by probing blots with an anti myc ab. The buffer that I used to elute is 0.1 M glycine (pH3) + myc peptide (20 micrograms/ml) again, many thanks in advance From Paul.Phelan from tufts.edu Fri Sep 4 11:21:29 2009 From: Paul.Phelan from tufts.edu (Paul J. Phelan) Date: Fri Sep 4 11:33:36 2009 Subject: protein purification problem In-Reply-To: References: Message-ID: <20090904122129.zgq3w2vhes4o8oco@webmail.tufts.edu> If I were you, I would answer a couple more questions first: When you say you express a lot of protein, is it right to assume that it is soluble, and you're not losing it (or much of it) in the insoluble material? It should be easy to tell if your protein is binding to your resin or not. Assay the flow-through from your column, or else take a sample of the resin (if you can) before and after adding your extract, boil the samples with SDS-PAGE loading buffer and run them on SDS-PAGE. I think it's critical to know if your protein is either not binding to your resin, or not coming off the resin, before trying to solve your problem. From martinseac from gmail.com Fri Sep 4 11:53:58 2009 From: martinseac from gmail.com (Emerson Martins) Date: Fri Sep 4 21:25:52 2009 Subject: cellular localization of L-arginine on confocal Message-ID: <8cccde200909040953y363f0478gbff2ac3b2d68e0d7@mail.gmail.com> Hi All I have to localize L-arginine in live cells on confocal microscopy. Does anyone know some fluorescent marker that I can use to treat cells to see L-arginine influx? Tks From engelbert_buxbaum from hotmail.com Tue Sep 8 08:50:41 2009 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Tue Sep 8 13:07:07 2009 Subject: cellular localization of L-arginine on confocal References: Message-ID: Am 04.09.2009, 12:53 Uhr, schrieb Emerson Martins : > Hi All > I have to localize L-arginine in live cells on confocal microscopy. Does > anyone know some fluorescent marker that I can use to treat cells to see > L-arginine influx? Arg can be labelled with reagents that carry a glyoxal-group (-CO-CO-). However, that's usually done with peptidyl-Arg, not the free amino acid. How is the Arg supposed to enter the cell? Can you use patch clamp or potential sensitive dyes? From martinseac from gmail.com Tue Sep 8 13:44:00 2009 From: martinseac from gmail.com (Emerson A. Castilho Martins) Date: Tue Sep 8 17:04:29 2009 Subject: cellular localization of L-arginine on confocal In-Reply-To: References: Message-ID: That's the problem. I want to label free L-arginine. I can use H3-L-arginine* to this, but I don't know a method to detect this accumulation by confocal microscopy, just by scintillography. Arg will enter the cell by cationic amino acids transporters, that was already described. Em Tue, 08 Sep 2009 10:50:41 -0300, Dr Engelbert Buxbaum escreveu: > Am 04.09.2009, 12:53 Uhr, schrieb Emerson Martins : > >> Hi All >> I have to localize L-arginine in live cells on confocal microscopy. Does >> anyone know some fluorescent marker that I can use to treat cells to see >> L-arginine influx? > > Arg can be labelled with reagents that carry a glyoxal-group (-CO-CO-). > However, that's usually done with peptidyl-Arg, not the free amino acid. > How is the Arg supposed to enter the cell? Can you use patch clamp or > potential sensitive dyes? > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods -- Usando o revolucion?rio cliente de e-mail do Opera: http://www.opera.com/mail/ From mathilde from sun.ac.za Wed Sep 9 07:39:45 2009 From: mathilde from sun.ac.za (Van der Merwe, M, Me ) Date: Wed Sep 9 10:32:27 2009 Subject: RNAseH Message-ID: <23F5B81FC6B6C24187F8122297646F6119FFB3126E@STBEVS08.stb.sun.ac.za> Hi all I am doing reverse transcription with Superscript II and oligo(dT)s as the first step of a 2-step RT qPCR. I will do relative quantification on fragments not exceeding 250 bp in size. How necessary is it to RNAseH treat my cDNA/RNA after reverse transcription? If I don't, will it cause trouble downstream? Thanks! Mathilde From nick.theodorakis from gmail.com Wed Sep 9 12:39:07 2009 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Wed Sep 9 13:41:27 2009 Subject: RNAseH References: Message-ID: On Sep 9, 7:39?am, "Van der Merwe, M, Me " wrote: > Hi all > > I am doing reverse transcription with Superscript II and oligo(dT)s as the first step of a 2-step RT qPCR. I will do relative quantification on fragments ?not exceeding 250 bp in size. > > How necessary is it to RNAseH treat my cDNA/RNA after reverse transcription? If I don't, will it cause trouble downstream? > > Thanks! > Mathilde I never do RNase H treatment for routine quantitative or semi- quantitative RT-PCR. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From vpireiner from gmail.com Wed Sep 9 14:29:55 2009 From: vpireiner from gmail.com (Becky Pickin) Date: Wed Sep 9 15:07:11 2009 Subject: RNAseH In-Reply-To: <23F5B81FC6B6C24187F8122297646F6119FFB3126E@STBEVS08.stb.sun.ac.za> References: <23F5B81FC6B6C24187F8122297646F6119FFB3126E@STBEVS08.stb.sun.ac.za> Message-ID: I always do an RNase treatment. I think it was not necessary for amplified genes isolated from transfected cells, but it does seem to be necessary when isolating RNA from non-transfected cells. I figure it can't hurt. If you are curious, you can always set up no-RT comparrison reactions and compare RNase treated versions of +/- RT reactions to check. ~RRP On Wed, Sep 9, 2009 at 8:39 AM, Van der Merwe, M, Me < mathilde@sun.ac.za> wrote: > Hi all > > I am doing reverse transcription with Superscript II and oligo(dT)s as the > first step of a 2-step RT qPCR. I will do relative quantification on > fragments not exceeding 250 bp in size. > > How necessary is it to RNAseH treat my cDNA/RNA after reverse > transcription? If I don't, will it cause trouble downstream? > > Thanks! > Mathilde > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From shifalich from rediffmail.com Thu Sep 10 08:58:37 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Sep 10 10:27:35 2009 Subject: RNAseH Message-ID: <1252510331.S.3121.17085.f4mail-234-246.rediffmail.com.old.1252591117.62195@webmail.rediffmail.com> I also don't do RNaseH treatment, I was told by company guys that RNA being an unstable molecule will get degraded by itself in the second step. Also, you can check the RT that you are using is RNase H + or negative. Hope this helps!!!!!!! Shifali On Wed, 09 Sep 2009 21:02:11 +0530 wrote >Hi all I am doing reverse transcription with Superscript II and oligo(dT)s as the first step of a 2-step RT qPCR. I will do relative quantification on fragments not exceeding 250 bp in size. How necessary is it to RNAseH treat my cDNA/RNA after reverse transcription? If I don't, will it cause trouble downstream? Thanks! Mathilde _______________________________________________ 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 Thu Sep 10 08:58:37 2009 From: shifalich from rediffmail.com (shifali chatrath) Date: Thu Sep 10 10:27:47 2009 Subject: RNAseH Message-ID: <1252510331.S.3121.17085.f4mail-234-246.rediffmail.com.old.1252591117.62195@webmail.rediffmail.com> I also don't do RNaseH treatment, I was told by company guys that RNA being an unstable molecule will get degraded by itself in the second step. Also, you can check the RT that you are using is RNase H + or negative. Hope this helps!!!!!!! Shifali On Wed, 09 Sep 2009 21:02:11 +0530 wrote >Hi all I am doing reverse transcription with Superscript II and oligo(dT)s as the first step of a 2-step RT qPCR. I will do relative quantification on fragments not exceeding 250 bp in size. How necessary is it to RNAseH treat my cDNA/RNA after reverse transcription? If I don't, will it cause trouble downstream? Thanks! Mathilde _______________________________________________ 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 blackhole from abuse.plus.com Thu Sep 10 10:51:50 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Thu Sep 10 12:01:23 2009 Subject: RNAseH References: Message-ID: <9kYCggAWCSqKFAX2@abuse.plus.com> Historians believe that in newspost on Wed, 9 Sep 2009, "Van der Merwe, M, Me " penned the following literary masterpiece: >How necessary is it to RNAseH treat my cDNA/RNA after reverse transcription? It isn't. > If I don't, will it cause trouble downstream? No. Probably not much RNA left after 95C to stop the reaction, or denature the DNA in the PCR, in the presence of Mg in the reaction buffer. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From gerchman from research.haifa.ac.il Thu Sep 10 14:54:58 2009 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Thu Sep 10 16:13:29 2009 Subject: pTopo purification problems Message-ID: <1252612498.4aa95992c8c3a@webmail.haifa.ac.il> Greetings netters I have recently made an attempt to clone a short gene into pTopo (using Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried already 2 different kits). Any ideas as to how remediate this situation? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From taliaferrod from mail.nih.gov Thu Sep 10 16:31:20 2009 From: taliaferrod from mail.nih.gov (Taliaferro, Dwayne (NIH/NIMH) [F]) Date: Thu Sep 10 20:12:38 2009 Subject: pTopo purification problems In-Reply-To: <1252612498.4aa95992c8c3a@webmail.haifa.ac.il> Message-ID: Um...try it again? On a constructive note... What size is the gene? Did you -tail it? Did you try a control reaction? Perhaps it was a plasmid prep issue? Are you sure the colony you picked was bacteria vs fungus? Are you sure your AMP is working? On 9/10/09 3:54 PM, "Yoram Gerchman" wrote: Greetings netters I have recently made an attempt to clone a short gene into pTopo (using Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried already 2 different kits). Any ideas as to how remediate this situation? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From bzahedi from bccrc.ca Thu Sep 10 18:48:19 2009 From: bzahedi from bccrc.ca (Bari Zahedi) Date: Thu Sep 10 20:12:45 2009 Subject: pTopo purification problems In-Reply-To: <26819_1252617551_1252617551_1252612498.4aa95992c8c3a@webmail.haifa.ac.il> References: <26819_1252617551_1252617551_1252612498.4aa95992c8c3a@webmail.haifa.ac.il> Message-ID: This is very little info. It could be your bugs, your lysis, your amp is off, it could be many things. Can you provide more info. To narrow down if the problem is at the plasmid prep level then: 1)To start, did you also get blue colonies? Can you get any plasmid out of those? This will help determine if there is something wrong with your kits/plasmid prep reagents. 2)Do you get anything coming out of your preps? Like a smear? If so the problem maybe with your lysis. -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Yoram Gerchman Sent: Thursday, September 10, 2009 12:55 PM To: methods@oat.bio.indiana.edu Subject: pTopo purification problems Greetings netters I have recently made an attempt to clone a short gene into pTopo (using Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried already 2 different kits). Any ideas as to how remediate this situation? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From cathalgarvey from gmail.com Fri Sep 11 02:33:04 2009 From: cathalgarvey from gmail.com (Cathal Garvey) Date: Fri Sep 11 10:05:57 2009 Subject: pTopo purification problems In-Reply-To: References: <1252612498.4aa95992c8c3a@webmail.haifa.ac.il> Message-ID: <468b1a400909110033g31e40763y66b03648714c47e9@mail.gmail.com> I recently had a similar problem, where after plasmid prep/enzyme cleavage, I would try to Gel Extract a fragment of the plasmid, only to find I had none left at the end. It turned out to be a bad Qiagen kit, it seems some of the buffers can cease to work right if they are left to age too long, even if sealed. If the pH on your buffers is wrong, you can expect to lose the DNA during preparation! Which might necessitate either some titration or a new kit. 2009/9/10 Taliaferro, Dwayne (NIH/NIMH) [F] > Um...try it again? > > On a constructive note... > > What size is the gene? > Did you -tail it? > Did you try a control reaction? > Perhaps it was a plasmid prep issue? > Are you sure the colony you picked was bacteria vs fungus? > Are you sure your AMP is working? > > On 9/10/09 3:54 PM, "Yoram Gerchman" > wrote: > > > > Greetings netters > I have recently made an attempt to clone a short gene into pTopo (using > Top10 > competent cells). i did get white colonies on LB-Amp-X-gal, and they grew > nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried > already 2 different kits). Any ideas as to how remediate this situation? > Many thanks > Yoram > > > > ------------------------------------------------------------------------ > This message was sent using IMP, the Webmail Program of Haifa University > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- letters.cunningprojects.com twitter.com/onetruecathal Kiva.org - Loans That Change Lives From blackhole from abuse.plus.com Fri Sep 11 07:09:20 2009 From: blackhole from abuse.plus.com (Duncan Clark) Date: Fri Sep 11 10:06:03 2009 Subject: pTopo purification problems References: Message-ID: Historians believe that in newspost on Thu, 10 Sep 2009, Yoram Gerchman penned the following literary masterpiece: >Any ideas as to how remediate this situation? Screen colonies by colony PCR using M13 forward and Reverse primers. Toothpick colony into 20ul water, swirl and grid on fresh plate using same toothpick. Heat kill 100C for 5mins then use 1ul into a PCR. 25-30cycles. No PCR product, assuming controls work, means no plasmid in those cells. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From virashkgupta from gmail.com Fri Sep 11 22:23:45 2009 From: virashkgupta from gmail.com (Virash Gupta) Date: Sat Sep 12 18:47:03 2009 Subject: Methods Digest, Vol 52, Issue 7 In-Reply-To: <200909111705.n8BH5Jp18072@net.bio.net> References: <200909111705.n8BH5Jp18072@net.bio.net> Message-ID: Besides all other answers, try this- If plasmid is there it will be isolated. If you using old solutions or old kit for plasmid purification- the main culprit is solution 2- alkaline lysis solution. It contains NaOH and as it gets repeatedel exposed to air-it absorbs carbon dioxide and converted to sod carbonate- to result in decreased alkalinity. The solution becomes inefficient in appropriate lysis. So prepare fresh solution and use. On 9/11/09, 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. pTopo purification problems (Yoram Gerchman) > 2. Re: pTopo purification problems > (Taliaferro, Dwayne (NIH/NIMH) [F]) > 3. RE: pTopo purification problems (Bari Zahedi) > 4. Re: pTopo purification problems (Duncan Clark) > 5. Re: pTopo purification problems (Cathal Garvey) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Thu, 10 Sep 2009 22:54:58 +0300 > From: Yoram Gerchman > Subject: pTopo purification problems > To: methods@oat.bio.indiana.edu > Message-ID: <1252612498.4aa95992c8c3a@webmail.haifa.ac.il> > Content-Type: text/plain; charset=windows-1255 > > > Greetings netters > I have recently made an attempt to clone a short gene into pTopo (using > Top10 > competent cells). i did get white colonies on LB-Amp-X-gal, and they grew > nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried > already 2 different kits). Any ideas as to how remediate this situation? > Many thanks > Yoram > > > > ------------------------------------------------------------------------ > This message was sent using IMP, the Webmail Program of Haifa University > > > > ------------------------------ > > Message: 2 > Date: Thu, 10 Sep 2009 17:31:20 -0400 > From: "Taliaferro, Dwayne (NIH/NIMH) [F]" > Subject: Re: pTopo purification problems > To: "methods@oat.bio.indiana.edu" > Message-ID: > > > Content-Type: text/plain; charset="iso-8859-1" > > Um...try it again? > > On a constructive note... > > What size is the gene? > Did you -tail it? > Did you try a control reaction? > Perhaps it was a plasmid prep issue? > Are you sure the colony you picked was bacteria vs fungus? > Are you sure your AMP is working? > > On 9/10/09 3:54 PM, "Yoram Gerchman" > wrote: > > > > Greetings netters > I have recently made an attempt to clone a short gene into pTopo (using > Top10 > competent cells). i did get white colonies on LB-Amp-X-gal, and they grew > nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried > already 2 different kits). Any ideas as to how remediate this situation? > Many thanks > Yoram > > > > ------------------------------------------------------------------------ > This message was sent using IMP, the Webmail Program of Haifa University > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > > > > ------------------------------ > > Message: 3 > Date: Thu, 10 Sep 2009 16:48:19 -0700 > From: Bari Zahedi > Subject: RE: pTopo purification problems > To: "'Yoram Gerchman'" , > "methods@oat.bio.indiana.edu" > Message-ID: > > Content-Type: text/plain; charset="us-ascii" > > This is very little info. It could be your bugs, your lysis, your amp is > off, it could be many things. Can you provide more info. > > To narrow down if the problem is at the plasmid prep level then: > 1)To start, did you also get blue colonies? Can you get any plasmid out of > those? This will help determine if there is something wrong with your > kits/plasmid prep reagents. > 2)Do you get anything coming out of your preps? Like a smear? If so the > problem maybe with your lysis. > > > -----Original Message----- > From: methods-bounces@oat.bio.indiana.edu [mailto: > methods-bounces@oat.bio.indiana.edu] On Behalf Of Yoram Gerchman > Sent: Thursday, September 10, 2009 12:55 PM > To: methods@oat.bio.indiana.edu > Subject: pTopo purification problems > > > Greetings netters > I have recently made an attempt to clone a short gene into pTopo (using > Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they > grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA > (tried already 2 different kits). Any ideas as to how remediate this > situation? > Many thanks > Yoram > > > > ------------------------------------------------------------------------ > This message was sent using IMP, the Webmail Program of Haifa University > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 4 > Date: Fri, 11 Sep 2009 13:09:20 +0100 > From: Duncan Clark > Subject: Re: pTopo purification problems > To: methods@net.bio.net > Message-ID: > Content-Type: text/plain;charset=us-ascii;format=flowed > > Historians believe that in newspost > on Thu, 10 Sep 2009, > Yoram Gerchman penned the following > literary masterpiece: > >Any ideas as to how remediate this situation? > > Screen colonies by colony PCR using M13 forward and Reverse primers. > > Toothpick colony into 20ul water, swirl and grid on fresh plate using > same toothpick. Heat kill 100C for 5mins then use 1ul into a PCR. > 25-30cycles. > > No PCR product, assuming controls work, means no plasmid in those cells. > > Duncan > -- > I love deadlines. I especially like the whooshing noise they make as > they go flying by. > > Duncan Clark > GeneSys Ltd. > > > ------------------------------ > > Message: 5 > Date: Fri, 11 Sep 2009 08:33:04 +0100 > From: Cathal Garvey > Subject: Re: pTopo purification problems > To: methods@oat.bio.indiana.edu > Message-ID: > <468b1a400909110033g31e40763y66b03648714c47e9@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > I recently had a similar problem, where after plasmid prep/enzyme cleavage, > I would try to Gel Extract a fragment of the plasmid, only to find I had > none left at the end. > > It turned out to be a bad Qiagen kit, it seems some of the buffers can > cease > to work right if they are left to age too long, even if sealed. If the pH > on > your buffers is wrong, you can expect to lose the DNA during preparation! > Which might necessitate either some titration or a new kit. > > 2009/9/10 Taliaferro, Dwayne (NIH/NIMH) [F] > > > Um...try it again? > > > > On a constructive note... > > > > What size is the gene? > > Did you -tail it? > > Did you try a control reaction? > > Perhaps it was a plasmid prep issue? > > Are you sure the colony you picked was bacteria vs fungus? > > Are you sure your AMP is working? > > > > On 9/10/09 3:54 PM, "Yoram Gerchman" > > wrote: > > > > > > > > Greetings netters > > I have recently made an attempt to clone a short gene into pTopo (using > > Top10 > > competent cells). i did get white colonies on LB-Amp-X-gal, and they grew > > nicely on liquid LB-Amp, but after plasmid purification I get no DNA > (tried > > already 2 different kits). Any ideas as to how remediate this situation? > > Many thanks > > Yoram > > > > > > > > ------------------------------------------------------------------------ > > This message was sent using IMP, the Webmail Program of Haifa University > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > letters.cunningprojects.com > twitter.com/onetruecathal > Kiva.org - Loans That Change Lives > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 52, Issue 7 > ************************************** > -- 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 vasu.nanobiotech91 from gmail.com Sat Sep 12 14:06:08 2009 From: vasu.nanobiotech91 from gmail.com (Shrinivas D) Date: Sat Sep 12 18:47:20 2009 Subject: Help in calculatiing Tm and GC content of primer Message-ID: <560e3e54-6414-4c09-bd49-e2a5efa2a231@u36g2000prn.googlegroups.com> Hi friends.. I am working with pET vector system. for expression of the enzme in E.coli BL21. I have to design primer with Nco1 site in forward primer. so can anybody will suggest me about the placing of ATG site of ORF...whether it should be in the primer's R.E site or else again i have to add in the primer aftre the R.E site. and one more thing for calculating the Tm and GC..in primer designing. whethet i should consider the R.E site sequence along with the primer sequence..for the calculation or else i should exclude it... waiting for suggestion from scientific community From cjmcdermottroe from googlemail.com Sun Sep 13 12:45:47 2009 From: cjmcdermottroe from googlemail.com (cjmcdermottroe@gmail.com) Date: Sun Sep 13 16:18:32 2009 Subject: pTopo purification problems Message-ID: <28751081.6.1252863950893.JavaMail.seven@ap1.p0.uk.7sys.net> Everybody has already suggested the obvious - dodgy amp/plasmid prep kit etc. I clone into topo more often that i'd like and dont bother with blue/white screening. Instead, i just use a no insert control plate as an indicator. Typically (for an average sized insert at a v:i ratio of 1:3 and ligation time of 5 min) i'll get approx 20 and 150 colonies on the no ins and the 1:3 plate, respectively. you ought to concentrate (again as already mentioned) on establishing whether or not your amp is off. Transform some bugs with a preprepared amp resistance plasmid that you or a colleague has in the lab. These should grow on amp agar whereas untransformed bugs should not. If this is the case, ensure the prep kit is ok by preping a colony or 2 from the above plate containing bugs that you transformed with your amp plasmid. Stick it on a gel (preferably linearised) and if you have a band, all is good. If not, buy a new miniprep kit. -- I sent this from my 3 mobile -- -original message- Subject: pTopo purification problems From: "Yoram Gerchman" Date: 10/09/2009 10:15 pm Greetings netters I have recently made an attempt to clone a short gene into pTopo (using Top10 competent cells). i did get white colonies on LB-Amp-X-gal, and they grew nicely on liquid LB-Amp, but after plasmid purification I get no DNA (tried already 2 different kits). Any ideas as to how remediate this situation? Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From eenigoy from gmail.com Wed Sep 16 08:29:09 2009 From: eenigoy from gmail.com (yoginee budhkar) Date: Wed Sep 16 11:52:17 2009 Subject: Primer designing for expression vector Message-ID: <77ddf6c70909160629w2728f07et62de421b3763acaf@mail.gmail.com> Dear All, I am planning to clone a gene of interest in qiagen expression system, using their vector pQE30 UA, in which directly the PCR amplified product can be cloned, just like the good old TA cloning. The design of primer should be in such a way that the start codon should form the first 3 nucleotide of the insert DNA. There can be no manipulations there! I have thus thought of taking 1st 23 nucleotides The problem is with the GC content of this primer. GC is just about 35%. I can manipulate the reverse primer since it is N terminal His tagging vector, therefore the ATG has to be in frame with the vector's other cis elements. Any warnings that I should be considering in particular? -- -- Yoginee Budhkar Senior Research Fellow Food Engineering and Technology Department Institute of Chemical Technology Matunga, Mumbai 400019 Contact: +91 9223355677 From martinseac from gmail.com Wed Sep 16 15:18:03 2009 From: martinseac from gmail.com (Emerson Martins) Date: Wed Sep 16 15:55:52 2009 Subject: Primer designing for expression vector In-Reply-To: <77ddf6c70909160629w2728f07et62de421b3763acaf@mail.gmail.com> References: <77ddf6c70909160629w2728f07et62de421b3763acaf@mail.gmail.com> Message-ID: <8cccde200909161318ua6fb1b1g5b5b55eb564da5d0@mail.gmail.com> I solved a similar problem cloning from a regiom upstream of the ORF, and then with the plasmid purified making a nested PCR with full ORF. It works... 2009/9/16 yoginee budhkar > Dear All, > > I am planning to clone a gene of interest in qiagen expression system, > using > their vector pQE30 UA, in which directly the PCR amplified product can be > cloned, just like the good old TA cloning. The design of primer should be > in > such a way that the start codon should form the first 3 nucleotide of the > insert DNA. There can be no manipulations there! > > I have thus thought of taking 1st 23 nucleotides The problem is with the GC > content of this primer. GC is just about 35%. I can manipulate the reverse > primer since it is N terminal His tagging vector, therefore the ATG has to > be in frame with the vector's other cis elements. > > Any warnings that I should be considering in particular? > > -- > -- > Yoginee Budhkar > Senior 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 anonwums1 from gmail.com Wed Sep 16 20:13:58 2009 From: anonwums1 from gmail.com (Adam .) Date: Thu Sep 17 09:58:20 2009 Subject: qRT-PCR on very few (10,000 - 20,000 cells) Message-ID: <858249120909161813s1490da23k975c1fc0a16704b2@mail.gmail.com> Hi all, I am attempting to do qRT-PCR using material from 10,000 - 20,000 cells. Here is what I'm doing: 1) FACS sort 10,000 - 20,000 cells into Trizol LS 2) Prepare RNA in Trizol LS per standard protocols 3) Make cDNA from the RNA using Invitrogen's SuperScript III First-Strand Synthesis System for RT-PCR 4) Perform qPCR on that cDNA using Applied Biosystem's RNA-to-Ct kit using Sybr green as a detector I have verified using 100,000 cells that I can amplify my target product. The problem is that when I go down to my actual samples of 10,000 cells, nothing amplifies, not even my housekeeping gene. I've seen people publish qRT-PCR on rare cell populations, but they rarely tell you how many cells they actually had to collect to get any usable data. Unfortunately for me, those 10,000 cells requires 5-10 transgenic mice and 5 hours of prep time, so simply increasing the cell number is probably not viable. Any suggestions on how to fix this? Do any of you routinely amplify such small amounts of RNA for qPCR or qRT-PCR? Do you amplify your RNA first using commercially available kits. If so, what kits? Thanks, Adam From as9n from virginia.edu Thu Sep 17 16:30:37 2009 From: as9n from virginia.edu (Ann Sutherland) Date: Thu Sep 17 18:23:12 2009 Subject: qRT-PCR on very few (10,000 - 20,000 cells) In-Reply-To: <200909171707.n8HH7up16704@net.bio.net> References: <200909171707.n8HH7up16704@net.bio.net> Message-ID: >Hi Adam, I have routinely done RT-PCR on samples of 100 mouse embryos at the 8-cell to blastocyst stage (so 800 to 6,000 cells total) with a wide variety of primers. I think you may not be quantitatively recovering your RNA in step 2. For large samples, Trizol works really well, but for small samples I think the percentage lost is too high. Instead of Trizol, I used the Micro-Fast Track kit from Invitrogen, and later the micro PolyA+ kit from Ambion to isolate polyA+ RNA from small samples and got great results. I also routinely use glycogen to co-precipitate with the RNA to get better yield. Ann >------------------------------ > >Hi all, > >I am attempting to do qRT-PCR using material from 10,000 - 20,000 cells. >Here is what I'm doing: > >1) FACS sort 10,000 - 20,000 cells into Trizol LS >2) Prepare RNA in Trizol LS per standard protocols >3) Make cDNA from the RNA using Invitrogen's SuperScript III First-Strand >Synthesis System for RT-PCR >4) Perform qPCR on that cDNA using Applied Biosystem's RNA-to-Ct kit using >Sybr green as a detector > >I have verified using 100,000 cells that I can amplify my target product. >The problem is that when I go down to my actual samples of 10,000 cells, >nothing amplifies, not even my housekeeping gene. I've seen people publish >qRT-PCR on rare cell populations, but they rarely tell you how many cells >they actually had to collect to get any usable data. Unfortunately for me, >those 10,000 cells requires 5-10 transgenic mice and 5 hours of prep time, >so simply increasing the cell number is probably not viable. > >Any suggestions on how to fix this? Do any of you routinely amplify such >small amounts of RNA for qPCR or qRT-PCR? Do you amplify your RNA first >using commercially available kits. If so, what kits? > >Thanks, >Adam > > >------------------------------ > >_______________________________________________ >Methods mailing list >Methods@net.bio.net >http://www.bio.net/biomail/listinfo/methods > >End of Methods Digest, Vol 52, Issue 11 >*************************************** -- Ann Sutherland Associate Professor Department of Cell Biology University of Virginia Health System 1300 Jefferson Park Ave. Jordan Hall 3-15 Charlottesville, VA 22908 ph: 434-243-6711 FAX: 434-982-3912 email: as9n@virginia.edu From lha from stowers.org Thu Sep 17 21:13:51 2009 From: lha from stowers.org (Zhan, Le) Date: Sun Sep 20 12:17:29 2009 Subject: RNA dot blot protocol? Message-ID: Hi Teresa This is Le Zhan, graduate student in Ron Yu lab from Stowers Institute of Medical Research in Kansas City,Missouri. I spot this RNA dot blot protocol question from online: http://www.bio.net/bionet/mm/methods/2006-December/101564.html The question was asked in 2006. And now, I have the same problem with my ISH probes or antibodies,and I would like to have a similar test of my probes and antibodies. So do you have a completed protocol for this purpose now? Thank you very much!! Le From Luke.Moertel from qimr.edu.au Mon Sep 21 21:20:19 2009 From: Luke.Moertel from qimr.edu.au (Luke Moertel) Date: Tue Sep 22 08:04:32 2009 Subject: Cot1 DNA preparation Message-ID: <4E51AF07E2E53D4C842526904C0FD26D056CC805@SPHINX.adqimr.ad.lan> Did you ever get a protocol for Cot-1 synthesis, I need it for a parasite Dr Luke Moertel BBSc PhD QIMR 300 Herston Road Herston 4006 Qld Australia From Priyesh.Rughani from nanoporetech.com Tue Sep 22 10:58:19 2009 From: Priyesh.Rughani from nanoporetech.com (Priyesh Rughani) Date: Tue Sep 22 11:37:12 2009 Subject: clean-up of ssDNA Message-ID: <6F9B8DAF2453CC438584662FB8ACA687043AC29F53@ontoxfex00> Dear Tom, A while ago you posted a thread (see below), I was wondering if you could share the link to the protocol you used for the generate ssDNA. I have searched the web, but cant not find the protocol you mentioned Thanks, Priyesh Rughani Dear Colleagues, I have isolated mg quantities of ssDNA following the protocol of Gruber et al in Focus 15: 59-65. I have a problem in that the OD 260/280 is about 1.5 after following the full protocol. I need to quantify this material precisely as well as having very clean ssDNA. Does anyone have any quick solutions for a cleanup of this large amount of material. In the protocol, multiple phenol-chloroform extractions have already been performed. If there is a FAQ to this point could someone direct me to it. Thanks, Tom Newman From dongocthuy73 from yahoo.com Tue Sep 22 11:11:42 2009 From: dongocthuy73 from yahoo.com (thuy dongoc) Date: Tue Sep 22 13:13:14 2009 Subject: LPS purfication Message-ID: <162853.46569.qm@web110601.mail.gq1.yahoo.com> Dear Sir/Madam I am looking for a protocol to isolate and purify LPS antigen from Salmonella. And I know that in one of the paragraph on the internet, you mentioned about a simple method which we can lyse the cells in 1% sarcosyl. ? http://www.bio.net/bionet/mm/methods/1995-June/029973.html ? It would be much appreciated if you could let me know in details about this method! Thank for your help. Regards, Thuy __________________________________________________________________________________ Get more done like never before with Yahoo!7 Mail. Learn more: http://au.overview.mail.yahoo.com/ From editor from gene-quantification.info Thu Sep 24 07:44:32 2009 From: editor from gene-quantification.info (Editor www.Gene-Quantification.info) Date: Thu Sep 24 07:52:20 2009 Subject: qPCR NEWS - September 2009 - key topic - circulating nucleic acids - CNA Message-ID: qPCR NEWS - September 2009 - key topic - circulating nucleic acids - CNA ---------------------------------------------------------------------------= ------------------- 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: - NEW PAGE - circulating nucleic acids =3D> http://CNA.gene-quantification.= info - a lot of updates in MIQE - media & press review =3D> http://miqe.gene-quantification.info/ - New molecular diagnostics qPCR/real-time PCR discussion forum: LinkedIN & XING - qPCR / real-time PCR BLOG =3D> http://real-time-pcr.blogspot.com/ - For better navigation we developed a TAG CLOUD =3D> http://directory.gene-quantification.info/ - New qPCR events in autumn 2009: symposia & workshops =3D> http://events.gene-quantification.info/ European wide qPCR application workshops - register now ! Course program autumn 2009 & winter 2010 =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf ---------------------------------------------------------------------------= ----- CNA =3D Circulating Nucleic Acids Most of the DNA and RNA in the body is located within cells, but a small amount of nucleic acids can also be found circulating freely in the blood. These DNA, RNA and small RNA molecules are thought to come from dying cells that release their contents into the blood as they break down. The term "Circulating Nucleic Acids =3D CNA" refers to segments of DNA or RNA found in the bloodstream. CNAs offers a non-invasive approach to a wide range in diagnostics of clinical disorders that will allow the basic information necessary not only for use in predictive medicine but also for direct use in acute medicine. Further free CNAs offer unique opportunities for early diagnosis of clinical conditions, e.g. in early cancer detection. Although DNA was first demonstrated in human blood from healthy donors, pregnant women and clinical patients in 1948, the structure of DNA was still to be determined as was the elucidation of its role as the basis of the gene. Consequently, no interest was shown in the presence of DNA in the circulatory system until high DNA levels were demonstrated in the blood of patients with systemic lupus erythematosus. Similar observations were also made in acute medicine, diabetes, oncology and fetal medicine. The presence of DNA and RNA in plasma of patients has been recognised since the 1970s. A range of markers have been proposed for the identification of a particular cancer, though there is frequent conflict in the literature as to the effectiveness of particular probes. However, recently, hypermethylated CpG in the promotor region of tumour suppressor genes has been suggested to trigger local gene silencing. Aberrant methylation of the p26 tumour suppressor gene was the first to be detected in liver, breast and lung cancer. Nucleic acids can be found in small amounts in healthy and diseased human plasma/serum. Higher concentrations of DNA are present in the plasma of cancer patients sharing some characteristics with DNA of tumor cells. Together with decreased strand stability, the presence of specific oncogene or tumor-suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes may be detected. Moreover, tumor-related mRNA has been found circulating in the plasma/serum. The results obtained in many different cancers have opened a new research area indicating that circulating nucleic acids might eventually be used for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer. For more info and for some key papers about circulating nucleic acids =3D> http://CNA.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 2010 in Vienna international qPCR Symposium 7-9th April 2010 Main Topic =93The ongoing evolution of qPCR=94 download updated 2nd announcement as PDF =3D> http://www.bioeps.com/qpcr2010/Vienna-qPCR-2010-2nd-announcement.pdf Symposium sessions & preliminary title MIQE and QM strategies in qPCR The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results. QM strategies in real- time PCR to guarantee better and more valid results. Prof. Stephen Bustin, =93The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments=94 High throughput quantitative PCR =96 digital PCR 384 well applications, new high throughput platforms, droplet PCR, qPCR robotics, digital PCR, gene expression real-time RT-PCR arrays (mRNA and microRNA), quantitative multiplexing, =85 Prof. Mikael Kubista, =93Digital PCR and intracellular expression profiling=94 Dr. Philip Day, =93High throughput droplet PCR=94 Dr. Ken Livak, "title to be announced" HRM =96 High Resolution Melting SNP analysis, HRM =3D high resolution melt applications, Epigenetics, methylation markers, HRM platform comparison, etc =85 Prof. Carl Wittwer, "High Resolution Melting Analysis" Prof. Claudio Orlando, =93High Resolution Melting Analysis in Cancer Diagnosis=94 Circulating nucleic acids Analysis of circulating RNAs and DNA and microRNAs as diagnostic and prognostic marker, =85 Dr. Pamela Pinzani, =93Cell free circulating DNA=94 Dr. Alfred Sch=F6ller, "Targeting the human urine RNAome for tumor diagnostics by qPCR=94 Single-cell qPCR Single-cell sampling, pre-amplification techniques, laser microdissection, sub-cellular PCR, micro-manipulation of cell clusters, cellular micro injection, FACS spotting, single cell handling, pre-amplification=85 Dr. Michael W. Pfaffl, =93Quantitative expression analysis after pre- amp in single WBCs=94 Dr. Anders Stahlberg, =93Single-cell gene expression profiling=94 RNAi - microRNA - siRNA Applications =96 miRNA normalisation RNAi mechanism, microRNA extraction, qRT-PCR technologies to detect microRNA, microRNA normalisation strategies, siRNA applications in combination with qRT-PCR, microRNA targets and microRNA precursors, new siRNA manipulation and microRNA technologies, ... Prof. Jo Vandesompele, =93MicroRNA and mRNA gene expression normalization=94 Dr. Mirco Castoldi, "Expression profiling of microRNA by quantitative real time PCR, what is available and where to go from there" 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, statistics in real-time PCR, data management, multiway expression profiling, multiple regression analysis, 3D data visualization, ... Dr. Ales Tichopad, =93Statistical aspects of quantitative PCR experiment design and qPCR data analysis=94 Dr. Jan Hellemans, =93Accurate and objective copy number profiling using real-time quantitative PCR=94 Dr. Anders Bergkvist, =93Expression profiling - clusters of possibilities=94 ---------------------------------------------------------------------------= ----- 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.) 19 - 23 October 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 26 - 28 October 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) 16 - 20 November 2009 (E) 3-day microRNA Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) 7 - 11 December 2009 (E) 3-day qPCR Core Module (Mon. - Wed.) & 2- day BioStatistics (Thu. - Fri.) =3D> http://www.gene-quantification.de/bioeps-courses-autumn-2009.pdf ---------------------------------------------------------------------------= ----- 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 sz2840 from gmail.com Fri Sep 25 23:21:12 2009 From: sz2840 from gmail.com (silin zhong) Date: Sat Sep 26 07:01:56 2009 Subject: can T7/SP6 transcribe eukaryotic DNA without bias Message-ID: Hi I am wondering whether one can convert large eukaryotic genomic DNA fragments to RNA. It is relatively easy to generate a promoter sequence, since adaptor containing T7 sequence (or other types of seq) can be easily ligated to gDNA fragments. Alternatively, PCR primers containing such promoter sequence can be used. I am not sure whether DNA regions with some secondary structure mimicking a T7/SP6 terminator will cause premature stop of the transcription hence no full-length transcript. If the RNA polymerases do have such a bias, genomic amplification kits with a DNA->RNA step will be problematic. cheers silin From shenq from purdue.edu Mon Sep 28 01:07:31 2009 From: shenq from purdue.edu (Qingwu Shen) Date: Mon Sep 28 07:41:16 2009 Subject: mitochondrial DNA copy number measurement In-Reply-To: References: <200909171707.n8HH7up16704@net.bio.net> Message-ID: <1254118051.4ac052a38d316@webmail.purdue.edu> Hi All, Does anybody have some experience with cell mitochondrial DNA copy number measurement that can share with me? I need to measure mtDNA copy number of sorted cells. So the sample cells could not be very high, with only 5,000- 10,000 cells. I need to isolate tatal DNA from cells first, and then use qPCR to amplify mtDNA. I have following questions that need your answer or suggestion: 1. Do you know any good kit for DNA isolation from such few cells? Also, the kit should compatible with PCR. 2. My sample cells are isolated from mouse skeletal muscle. Do you know where to find the mtDNA sequence for primer and probe design? I went to NCBI and just found a couple of mouse mitochondrial genes. It's better if I can find the whole mtDNA sequence. 3. Is there any difference in mitochondrial gene seqence between tissues? 4. This is also my first time working with qPCR. Could you please give me as detailed information as possible about primer and probe design and ordering, that to say, how to design and order the primers and probe. 5. Any suggestion about qPCR is more than welcome. Thanks, qs