From nikhil.samar from gmail.com Thu May 1 06:35:15 2008 From: nikhil.samar from gmail.com (Niksam) Date: Thu May 1 10:34:06 2008 Subject: LNA transfection! References: <47f36fd0$0$12642$3b214f66@aconews.univie.ac.at> Message-ID: <03ca0431-ae40-4249-b3ed-216c6a120bc0@m73g2000hsh.googlegroups.com> On Apr 2, 4:36?pm, "Andreas Ploner" wrote: > Hi everyone! > I've got a problem, maybe one of can help me. I want to transfect Locked > nucleic acids (LNAs) into 293T cells. HAs anyone of you expereince with LNA > transfection and whch transfection reagent do you use? Most publication use > Lipofectamin. > > At the moment im using Metafectene (www.biontex.com). At the moment it seems > that knock down somehow works, but I can't find out easily how efficient > LNAs get transfected- so I'd need some transfection reagent to comapre it > to! > > Thanks for your answers! > > Andi Try using invitrogen's RNAiMax or lipofectamine 2000. It produces better results. Best, Nikhil From shirisau from hotmail.com Thu May 1 12:40:27 2008 From: shirisau from hotmail.com (shiri shiri) Date: Thu May 1 15:42:16 2008 Subject: salmon sperm DNA and Northern again Message-ID: Hello, You posted once on a problem you had with your northern hybridization. As you posted, I experience the same problem, unspecific binding all over the membrane, even in a blank one. I changed all my compounds, increased the concentration of the blocking solution but nothing works... The weird thing it worked great for many years but now no one in our lab, even ones who were previously successful in it, cannot solve this issue... I was wondering if you had a chance to solve this problem???? I would appreciate any help/suggestion THANKS SHIRI From bmacgreg from unc.edu Fri May 2 13:05:05 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Fri May 2 18:52:44 2008 Subject: Salmon sperm DNA and Northern again In-Reply-To: <200805021706.m42H6KQ01798@net.bio.net> References: <200805021706.m42H6KQ01798@net.bio.net> Message-ID: <15F271AA-1F1A-4592-8B7E-005EAC67F7AF@unc.edu> Hi, We had that happen once, and in the end the only thing that helped was switching membrane manufacturers. The company swore they hadn't changed anything, but neither had we, so. Maybe you can get free samples from a couple of different companies? Barbara MacGregor On May 2, 2008, at 1:06 PM, 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. salmon sperm DNA and Northern again (shiri shiri) > > From: shiri shiri > Date: May 1, 2008 1:40:27 PM EDT > To: > Subject: salmon sperm DNA and Northern again > > > Hello, > > You posted once on a problem you had with your northern hybridization. > As you posted, I experience the same problem, unspecific binding > all over > the membrane, even in a blank one. I changed all my compounds, > increased the > concentration of the blocking solution but nothing works... > The weird thing it worked great for many years but now no one in > our lab, > even ones who were previously successful in it, cannot solve this > issue... > > I was wondering if you had a chance to solve this problem???? > > I would appreciate any help/suggestion > > THANKS > SHIRI > > > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From peter.ianakiev from gmail.com Fri May 2 15:31:14 2008 From: peter.ianakiev from gmail.com (peter) Date: Fri May 2 18:52:50 2008 Subject: EmulsionPCR Message-ID: Hi group, I need to make emulsion in the emulsionPCR uniform. Any suggestions? Thanks, Peter From yansun2005 from gmail.com Fri May 2 14:50:48 2008 From: yansun2005 from gmail.com (yan sun) Date: Fri May 2 18:53:00 2008 Subject: ethanol precipitation Message-ID: Dear all, I did spin label DNA experiment. I mixed my modified DNA with spin label material in DMF and fomamide solution. I get 100% label efficiency. (the analytical gel shows that there is only one spin label DNA band). I was wondering how to purify my spin labeled DNA? My other site spin labeled DNAs are not 100% spin label efficiency, so I RUN big PAGE gel, and use elutor to purified my target spin labeled DNA. during this process, I lose quite amount material. that is why I don't want to run big gel this time. I made a research. looks like ethanol precipitation works. anyone have experience, please feel free to share. option 2. can I use freezing dry method? Thx a lot!!! -- Y. F.S. From hyrax3 from gmail.com Sat May 3 07:47:48 2008 From: hyrax3 from gmail.com (Sarah) Date: Sat May 3 11:02:57 2008 Subject: Cb5R Assay Message-ID: Hello, I am looking for an assay to determine the activity of cytochrome b5 reductase in blood. Can anyone help? Thank you. Sarah From gbangera from bcc.ctc.edu Sun May 4 15:04:48 2008 From: gbangera from bcc.ctc.edu (Gita Bangera) Date: Sun May 4 21:03:17 2008 Subject: methylene blue in the DNA sample Message-ID: Hello, Has anyone tried adding methylene blue to their DNA sample before running it on the gel? Since methylene blue stains DNA, I am wondering if that would work. I am not interested in cutting out the bands from this gel or using the DNA in any way. I just want my students to be able to visualize the DNA on the gel and I really don't want to have to deal with EtBr. Thank you for your time. Gita Gita Bangera Ph.D. Instructor, Life Sciences (Biology) Bellevue Community College Tel: 425-564-4031 Fax: 425-564-4125 From maximilianh from gmail.com Mon May 5 04:44:30 2008 From: maximilianh from gmail.com (Maximilian Haeussler) Date: Mon May 5 10:23:37 2008 Subject: methylene blue in the DNA sample In-Reply-To: References: Message-ID: <76f031ae0805050244g73dd3f4bk86a23d9a7f85081c@mail.gmail.com> nick from biteisizebio wrote about these "safe" stains recently, also calculating cost per 100ml: http://bitesizebio.com/2008/03/03/ethidium-bromide-the-alternatives/ he writes that methylene blue is "Post strain only, in 0.025% (w/v) methylene blue in water." and according to him only bands above 500ng are visible. methylene blue is also part of those "run your DNA at home" instructions: http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/ cheers Max 2008/5/4 Gita Bangera : > Hello, > > > > Has anyone tried adding methylene blue to their DNA sample before > running it on the gel? Since methylene blue stains DNA, I am wondering > if that would work. I am not interested in cutting out the bands from > this gel or using the DNA in any way. I just want my students to be able > to visualize the DNA on the gel and I really don't want to have to deal > with EtBr. > > > > Thank you for your time. > > > > Gita > > > > Gita Bangera Ph.D. > > Instructor, Life Sciences (Biology) > > Bellevue Community College > > Tel: 425-564-4031 > > Fax: 425-564-4125 > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Maximilian Haeussler, tel: +33 6 12 82 76 16 From wgrus from umich.edu Mon May 5 13:19:41 2008 From: wgrus from umich.edu (Wendy Grus) Date: Mon May 5 13:26:05 2008 Subject: co-transfecting with multiple vectors Message-ID: <9d2782af0805051119l17782972h1524542fee60b728@mail.gmail.com> Hi all- I am transfecting some HEK 293 T cells to transiently express a few different genes. Some of these genes are on pcDNA3.1 vectors while some are on pCI vectors. Will having them on different vectors affect their expression levels? Will I have to play with the concentrations of each? Should I put all the genes in one vector (singly)? Thanks, Wendy Grus From frist from cc.umanitoba.ca Mon May 5 17:41:47 2008 From: frist from cc.umanitoba.ca (Brian Fristensky) Date: Mon May 5 19:11:28 2008 Subject: Bioinformatics Course: Vancouver area Message-ID: <200805052241.m45MflnP025056@mira.cc.umanitoba.ca> --------------------------------------------------- Genome Canada APPLIED COMPUTATIONAL GENOMICS COURSE Simon Fraser University, Burnaby, BC, Canada July 24 - 30, 2008 --------------------------------------------------- The ACGC introduces biologists to some of the latest methods in bioinformatics, including DNA and protein sequence analysis, automation of data analysis, web services and workflows, and high-throughput genome annotation. The course is led by researchers from the Genome Canada Bioinformatics Platform. Both lectures and extensive hands-on tutorial sessions are included. For full details see: For more information visit the website: http://www.gcbioinformatics.ca/training ________________________________________________________________________________ The project is supported by Genome Alberta and Genome Canada, a not-for-profit organization which is leading Canada's national strategy on genomics with $ 600 million in funding from the federal government. From gparisi from farmbiol.uniba.it Tue May 6 03:39:33 2008 From: gparisi from farmbiol.uniba.it (Giovanni Parisi) Date: Tue May 6 10:33:23 2008 Subject: transfecting cells with viral particles Message-ID: <5d99051717c003bcfba1fa9109b758e4@farmbiol.uniba.it> hi all, I am trying to transfect some fibroblasts and HEK 293 using viruses I produced that transiently express shRNA. This is a tecnique never used in my laboratory so I have not methods to tritate viruses so I am using different volumes of obtained viruses. The first silencing experiment was done using viral particles obtained from SIGMA and I had some silencing but last time I had not silencing with viruses particles I produced. Someone told me that producing viral particles with mission system from SIGMA using HEK 293T cells is a very efficent process. Is it possible that am I using a low concentration of viruses? The second question is the following: I have intention to modify lentiviral vector pLKO1 replaceing U6 promoter with CMV promoter and cloning GFP cDNA in way to transfect cells to control efficiency of transfection. Is it possible to do it? thanx From peter.ianakiev from gmail.com Tue May 6 07:52:21 2008 From: peter.ianakiev from gmail.com (peter) Date: Tue May 6 10:33:30 2008 Subject: EmulsionPCR References: Message-ID: On May 2, 4:31 pm, peter wrote: > Hi group, I need to make emulsion in the emulsionPCR uniform. Any > suggestions? > Thanks, > Peter What is going on? No experts in emulsion PCR on this board? No one reads it? Anyway good luck with your research From morera98 from gmail.com Wed May 7 21:24:27 2008 From: morera98 from gmail.com (juan pablo morera rojas) Date: Thu May 8 10:37:36 2008 Subject: Question Message-ID: <887f1c940805071924p7fc7d27dn5ef4575755522b41@mail.gmail.com> Hi, my name is Juan Pablo Morera, Im from Costa Rica. Im sorry if Im bothering you in any way. I wanted to ask you about one primer Im working with and if you could help me. It has 52bp but I dont know why is so long, I found another paper that includes a shorter primer but still I have the longer one (it works by the way) besides it includes a enzyme restriction site, just adding an "A" in the end of the primer, but originally in the sequence there isnt site for any enzyme. If you have some clue about it I would really appreciate it. PSD I just know that it works but I dont know how does it. Thank you very much for your time and Attention. Juan Pablo Morera From sudhee26 from gmail.com Thu May 8 09:31:10 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 8 10:37:52 2008 Subject: Luciferase Assay! Message-ID: Hi all, 1. I am doing Luciferase assay to understand transcriptional regulation. I was looking for proper reporter lysis buffers. i found out one with KH2PO4 (7.8 pH) with 0.2% Triton X-100 (v/v)..is there any better? it takes time for us to order promega 5x reporter lysis buffer. 2. i am doing duplicate assays in 6 well plates..and collecting cells in pbs and lysing them in 200uL buffer. however i am not sure what is the "standard" lysate:luciferase substrate (roche) proportion. i have tried 10uL:90uL, 40uL:60uL, 100:100uL combinations, later gives me better RLUs we use a stand-alone, tube-luminometer (sirius), and do not use injectors. please let me know. (i will post my questions on DLuciferase Assay also..soon :)) thanks in advance sudheendra. -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Thu May 8 09:43:51 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 8 10:37:58 2008 Subject: EmulsionPCR In-Reply-To: References: Message-ID: i thought, vortexing itself would make the emulsion uniform On Tue, May 6, 2008 at 6:22 PM, peter wrote: > On May 2, 4:31 pm, peter wrote: > > Hi group, I need to make emulsion in the emulsionPCR uniform. Any > > suggestions? > > Thanks, > > Peter > > What is going on? No experts in emulsion PCR on this board? No one > reads it? > Anyway good luck with your research > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Thu May 8 09:49:05 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 8 10:38:04 2008 Subject: co-transfecting with multiple vectors In-Reply-To: <9d2782af0805051119l17782972h1524542fee60b728@mail.gmail.com> References: <9d2782af0805051119l17782972h1524542fee60b728@mail.gmail.com> Message-ID: Hey, look in to some of these http://www.springerlink.com/content/u3531825942t18v5/ http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html best, sudheendra. On Mon, May 5, 2008 at 11:49 PM, Wendy Grus wrote: > Hi all- > I am transfecting some HEK 293 T cells to transiently express a few > different genes. Some of these genes are on pcDNA3.1 vectors while some > are > on pCI vectors. Will having them on different vectors affect their > expression levels? Will I have to play with the concentrations of each? > Should I put all the genes in one vector (singly)? > > Thanks, > Wendy Grus > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Thu May 8 09:53:55 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 8 10:38:10 2008 Subject: co-transfecting with multiple vectors In-Reply-To: References: <9d2782af0805051119l17782972h1524542fee60b728@mail.gmail.com> Message-ID: and this! http://www.protocol-online.org/biology-forums/posts/25380.html i have done triple transient transfections..but yes..cell death is little more than single/double. But i seed more cells than i usually for single/double..and it seems to work.. plasmid expression can be altered depending on promoter sequence..especially multiple plasmid with different promoters..i try keeping them constant (CMV-all CMV, that way) best, sudheendra. On Thu, May 8, 2008 at 8:19 PM, Sudheendra Rao N R wrote: > Hey, look in to some of these > http://www.springerlink.com/content/u3531825942t18v5/ > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html > > best, > sudheendra. > > On Mon, May 5, 2008 at 11:49 PM, Wendy Grus wrote: > > > Hi all- > > I am transfecting some HEK 293 T cells to transiently express a few > > different genes. Some of these genes are on pcDNA3.1 vectors while some > > are > > on pCI vectors. Will having them on different vectors affect their > > expression levels? Will I have to play with the concentrations of each? > > Should I put all the genes in one vector (singly)? > > > > Thanks, > > Wendy Grus > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Thu May 8 10:14:05 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 8 10:38:18 2008 Subject: Does Lipofectamine cause gene knock down? In-Reply-To: <31DC2DFFD70C174B8E3072803F894F91046C71@EVSSTAFF1.livad.liv.ac.uk> References: <31DC2DFFD70C174B8E3072803F894F91046C71@EVSSTAFF1.livad.liv.ac.uk> Message-ID: Hi, quite scary i must say :) but if i am getting u right, u are trying to knock-down a gene using siRNA / mock siRNA in a stably transfected cell line(parental), which is also ur control cell line (which u are treating with only lipofectamine) knockdown is more in control cell line!! than proper siRNA actually knockdown is present in all transfected cell lines!! your control cell line no longer a control cell line. any contamination in lifofectamine? or optiMEM? try chaning the tranfection agent.. On Thu, Apr 24, 2008 at 2:43 PM, Kamalian, Laleh wrote: > Hi everyone > I have come across this problem. I have stably transfected my cells using > Lipofectamine 2000 as the transfection reagent. So far, I have been using > my parental cells as the calibrator during transient transfection. Now for > the stable transfection I have used scrambled si RNA oligonucleotide to > transfect my cells and used a pool of these cells as my control. Using > quantitative PCR to measure the mRNA of the colony cells and the control > cells, my colony cells show about 70% gene knock down comparing to the > scrambled si RNA control cells, which is good. However comparing to the > parental cells, the control shows about 90% less expression of the gene of > interest. Once before during my transient transfection I had the same > problem when used "no DNA" as my negative control i.e. Lipofectamine without > any DNA. At that time I had used western blot to measure the protein > instead of QPCR. I had noticed that my negative control lysate had > considerable decrease in protein expression. My question now is can > Lipofectamine or transfection condition cause the gene knock down? It > especially doesn't make sense now that my scrambled RNA control cells have > been growing in the selective medial for at least 4 weeks now. I am > thinking that even if the transfection condition had affected the cells > transiently, it can't effect the mRNA level after being eliminated from the > growth media. Am I right or am I missing something in here? > Laleh. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From tk from mit.edu Thu May 8 12:22:47 2008 From: tk from mit.edu (Tom Knight) Date: Fri May 9 10:49:07 2008 Subject: Question References: Message-ID: "juan pablo morera rojas" writes: > I wanted to ask you about one primer Im working > with and if you could help me. It has 52bp but I dont know why is so long, I > found another paper that includes a shorter primer but still I have > the longer one (it works by the way) besides it includes a enzyme > restriction site, just adding an "A" in the end of the primer, but > originally in the sequence there isnt site for any enzyme. If you have some > clue about it I would really appreciate it. It is quite common to add additional bases at the 5' end of a primer which do not match the DNA sequence being amplified. These extra bases are incorporated into the PCR product when the complementary strand is copied. Likely the last 18-25 bp of your primer will match the region of template DNA you are amplifying. That is all that is required for amplification. The remaining bases will simply be tagged onto the product at each end, which allows you to design cloning sites or other structure into the ends of the PCR product -- a very powerful, general technique. From jyyoungjimmy from gmail.com Sat May 10 16:38:43 2008 From: jyyoungjimmy from gmail.com (Jim Young) Date: Sun May 11 12:43:48 2008 Subject: Methods Digest, Vol 36, Issue 4 In-Reply-To: <200805051705.m45H5IQ25232@net.bio.net> References: <200805051705.m45H5IQ25232@net.bio.net> Message-ID: <57f211620805101438w495749ean678019458b891910@mail.gmail.com> That is simple. Our lab is using this pre-cast gel product, No EB, with prestain DNA marker. About $40 for 10 gels and ready to use, very nice. http://www.genscript.com/agarose_gel_kits.html On Mon, May 5, 2008 at 1:05 PM, 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. methylene blue in the DNA sample (Gita Bangera) > 2. Re: methylene blue in the DNA sample (Maximilian Haeussler) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Sun, 4 May 2008 13:04:48 -0700 > From: "Gita Bangera" > Subject: methylene blue in the DNA sample > To: > Message-ID: > > Content-Type: text/plain; charset="us-ascii" > > Hello, > > > > Has anyone tried adding methylene blue to their DNA sample before > running it on the gel? Since methylene blue stains DNA, I am wondering > if that would work. I am not interested in cutting out the bands from > this gel or using the DNA in any way. I just want my students to be able > to visualize the DNA on the gel and I really don't want to have to deal > with EtBr. > > > > Thank you for your time. > > > > Gita > > > > Gita Bangera Ph.D. > > Instructor, Life Sciences (Biology) > > Bellevue Community College > > Tel: 425-564-4031 > > Fax: 425-564-4125 > > > > > > ------------------------------ > > Message: 2 > Date: Mon, 5 May 2008 11:44:30 +0200 > From: "Maximilian Haeussler" > Subject: Re: methylene blue in the DNA sample > To: "Gita Bangera" > Cc: methods@magpie.bio.indiana.edu > Message-ID: > <76f031ae0805050244g73dd3f4bk86a23d9a7f85081c@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > nick from biteisizebio wrote about these "safe" stains recently, also > calculating cost per 100ml: > http://bitesizebio.com/2008/03/03/ethidium-bromide-the-alternatives/ > > he writes that methylene blue is "Post strain only, in 0.025% (w/v) > methylene blue in water." and according to him only bands above 500ng > are visible. > > methylene blue is also part of those "run your DNA at home" instructions: > > http://www.scq.ubc.ca/the-macgyver-project-genomic-dna-extraction-and-gel-electrophoresis-experiments-using-everyday-materials/ > > cheers > Max > > 2008/5/4 Gita Bangera : > > Hello, > > > > > > > > Has anyone tried adding methylene blue to their DNA sample before > > running it on the gel? Since methylene blue stains DNA, I am wondering > > if that would work. I am not interested in cutting out the bands from > > this gel or using the DNA in any way. I just want my students to be able > > to visualize the DNA on the gel and I really don't want to have to deal > > with EtBr. > > > > > > > > Thank you for your time. > > > > > > > > Gita > > > > > > > > Gita Bangera Ph.D. > > > > Instructor, Life Sciences (Biology) > > > > Bellevue Community College > > > > Tel: 425-564-4031 > > > > Fax: 425-564-4125 > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Maximilian Haeussler, > tel: +33 6 12 82 76 16 > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 36, Issue 4 > ************************************** > From jyyoungjimmy from gmail.com Sat May 10 16:53:55 2008 From: jyyoungjimmy from gmail.com (Jim Young) Date: Sun May 11 12:44:11 2008 Subject: Methods Digest, Vol 36, Issue 6 In-Reply-To: <200805081706.m48H69O28757@net.bio.net> References: <200805081706.m48H69O28757@net.bio.net> Message-ID: <57f211620805101453v7031d205l5857301333861a9c@mail.gmail.com> I have done similar knock-down. I am sure that bad transfection will cause a lot of problems. Why do you use si RNA oligonucleotide instead of vectors. You can have a siRNA expression vector to express siRNA for you. The results are much better. You can see the links for details: http://www.genscript.com/targeting_vector_construction.html http://www.genscript.com/siRNA_service.html On Thu, May 8, 2008 at 1:06 PM, 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. Question (juan pablo morera rojas) > 2. Luciferase Assay! (Sudheendra Rao N R) > 3. Re: EmulsionPCR (Sudheendra Rao N R) > 4. Re: co-transfecting with multiple vectors (Sudheendra Rao N R) > 5. Re: co-transfecting with multiple vectors (Sudheendra Rao N R) > 6. Re: Does Lipofectamine cause gene knock down? (Sudheendra Rao N R) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 7 May 2008 20:24:27 -0600 > From: "juan pablo morera rojas" > Subject: Question > To: "Huang Ke-xue" > Message-ID: > <887f1c940805071924p7fc7d27dn5ef4575755522b41@mail.gmail.com> > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, my name is Juan Pablo Morera, Im from Costa Rica. Im sorry if Im > bothering you in any way. I wanted to ask you about one primer Im working > with and if you could help me. It has 52bp but I dont know why is so long, > I > found another paper that includes a shorter primer but still I have > the longer one (it works by the way) besides it includes a enzyme > restriction site, just adding an "A" in the end of the primer, but > originally in the sequence there isnt site for any enzyme. If you have some > clue about it I would really appreciate it. > > PSD I just know that it works but I dont know how does it. > Thank you very much for your time and Attention. > > Juan Pablo Morera > > > ------------------------------ > > Message: 2 > Date: Thu, 8 May 2008 20:01:10 +0530 > From: "Sudheendra Rao N R" > Subject: Luciferase Assay! > To: methods , > methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi all, > 1. I am doing Luciferase assay to understand transcriptional regulation. I > was looking for proper reporter lysis buffers. > i found out one with KH2PO4 (7.8 pH) with 0.2% Triton X-100 (v/v)..is there > any better? > it takes time for us to order promega 5x reporter lysis buffer. > > 2. i am doing duplicate assays in 6 well plates..and collecting cells in > pbs > and lysing them in 200uL buffer. however i am not sure what is the > "standard" lysate:luciferase substrate (roche) proportion. > i have tried 10uL:90uL, 40uL:60uL, 100:100uL combinations, later gives me > better RLUs > we use a stand-alone, tube-luminometer (sirius), and do not use injectors. > > please let me know. > (i will post my questions on DLuciferase Assay also..soon :)) > > thanks in advance > sudheendra. > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > Message: 3 > Date: Thu, 8 May 2008 20:13:51 +0530 > From: "Sudheendra Rao N R" > Subject: Re: EmulsionPCR > To: peter , methods > > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > i thought, > vortexing itself would make the emulsion uniform > > On Tue, May 6, 2008 at 6:22 PM, peter wrote: > > > On May 2, 4:31 pm, peter wrote: > > > Hi group, I need to make emulsion in the emulsionPCR uniform. Any > > > suggestions? > > > Thanks, > > > Peter > > > > What is going on? No experts in emulsion PCR on this board? No one > > reads it? > > Anyway good luck with your research > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > Message: 4 > Date: Thu, 8 May 2008 20:19:05 +0530 > From: "Sudheendra Rao N R" > Subject: Re: co-transfecting with multiple vectors > To: "Wendy Grus" , methods > , > methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hey, look in to some of these > http://www.springerlink.com/content/u3531825942t18v5/ > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html > > best, > sudheendra. > > On Mon, May 5, 2008 at 11:49 PM, Wendy Grus wrote: > > > Hi all- > > I am transfecting some HEK 293 T cells to transiently express a few > > different genes. Some of these genes are on pcDNA3.1 vectors while some > > are > > on pCI vectors. Will having them on different vectors affect their > > expression levels? Will I have to play with the concentrations of each? > > Should I put all the genes in one vector (singly)? > > > > Thanks, > > Wendy Grus > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > Message: 5 > Date: Thu, 8 May 2008 20:23:55 +0530 > From: "Sudheendra Rao N R" > Subject: Re: co-transfecting with multiple vectors > To: "Wendy Grus" , methods > , > methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > and this! > http://www.protocol-online.org/biology-forums/posts/25380.html > i have done triple transient transfections..but yes..cell death is little > more than single/double. But i seed more cells than i usually for > single/double..and it seems to work.. > plasmid expression can be altered depending on promoter > sequence..especially > multiple plasmid with different promoters..i try keeping them constant > (CMV-all CMV, that way) > > best, > sudheendra. > On Thu, May 8, 2008 at 8:19 PM, Sudheendra Rao N R > wrote: > > > Hey, look in to some of these > > http://www.springerlink.com/content/u3531825942t18v5/ > > > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090500.html > > > > http://www.bio.net/hypermail/methds-reagnts/2001-September/090499.html > > > > best, > > sudheendra. > > > > On Mon, May 5, 2008 at 11:49 PM, Wendy Grus wrote: > > > > > Hi all- > > > I am transfecting some HEK 293 T cells to transiently express a few > > > different genes. Some of these genes are on pcDNA3.1 vectors while > some > > > are > > > on pCI vectors. Will having them on different vectors affect their > > > expression levels? Will I have to play with the concentrations of > each? > > > Should I put all the genes in one vector (singly)? > > > > > > Thanks, > > > Wendy Grus > > > _______________________________________________ > > > Methods mailing list > > > Methods@net.bio.net > > > http://www.bio.net/biomail/listinfo/methods > > > > > > > > > > > -- > > Think before agree > > Think before you nod > > but STOP thinking > > and You Are God > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > Message: 6 > Date: Thu, 8 May 2008 20:44:05 +0530 > From: "Sudheendra Rao N R" > Subject: Re: Does Lipofectamine cause gene knock down? > To: "Kamalian, Laleh" , methods > , methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi, > quite scary i must say :) > but if i am getting u right, u are trying to knock-down a gene using siRNA > / > mock siRNA in a stably transfected cell line(parental), which is also ur > control cell line (which u are treating with only lipofectamine) > > knockdown is more in control cell line!! than proper siRNA > actually knockdown is present in all transfected cell lines!! > > your control cell line no longer a control cell line. > any contamination in lifofectamine? or optiMEM? > try chaning the tranfection agent.. > > > > > On Thu, Apr 24, 2008 at 2:43 PM, Kamalian, Laleh < > L.Kamalian@liverpool.ac.uk> > wrote: > > > Hi everyone > > I have come across this problem. I have stably transfected my cells > using > > Lipofectamine 2000 as the transfection reagent. So far, I have been > using > > my parental cells as the calibrator during transient transfection. Now > for > > the stable transfection I have used scrambled si RNA oligonucleotide to > > transfect my cells and used a pool of these cells as my control. Using > > quantitative PCR to measure the mRNA of the colony cells and the control > > cells, my colony cells show about 70% gene knock down comparing to the > > scrambled si RNA control cells, which is good. However comparing to the > > parental cells, the control shows about 90% less expression of the gene > of > > interest. Once before during my transient transfection I had the same > > problem when used "no DNA" as my negative control i.e. Lipofectamine > without > > any DNA. At that time I had used western blot to measure the protein > > instead of QPCR. I had noticed that my negative control lysate had > > considerable decrease in protein expression. My question now is can > > Lipofectamine or transfection condition cause the gene knock down? It > > especially doesn't make sense now that my scrambled RNA control cells > have > > been growing in the selective medial for at least 4 weeks now. I am > > thinking that even if the transfection condition had affected the cells > > transiently, it can't effect the mRNA level after being eliminated from > the > > growth media. Am I right or am I missing something in here? > > Laleh. > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > -- > Think before agree > Think before you nod > but STOP thinking > and You Are God > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 36, Issue 6 > ************************************** > From jyyoungjimmy from gmail.com Sat May 10 16:32:11 2008 From: jyyoungjimmy from gmail.com (Jim Young) Date: Sun May 11 12:44:17 2008 Subject: Methods Digest, Vol 36, Issue 3 In-Reply-To: <200805031705.m43H5uQ06403@net.bio.net> References: <200805031705.m43H5uQ06403@net.bio.net> Message-ID: <57f211620805101432m402c0ea2u32a1b7e63c424525@mail.gmail.com> About the DNA purification problem, you may use glycogen to purify your DNA. You may google glycogen for details. It is so easy. By the way, I used the products and services from http://www.genscript.com/. Their PCR products are not expensive and pretty good. On Sat, May 3, 2008 at 1:05 PM, 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: Salmon sperm DNA and Northern again (Barbara MacGregor) > 2. EmulsionPCR (peter) > 3. ethanol precipitation (yan sun) > 4. Cb5R Assay (Sarah) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Fri, 2 May 2008 14:05:05 -0400 > From: Barbara MacGregor > Subject: Re: Salmon sperm DNA and Northern again > To: methods@oat.bio.indiana.edu > Message-ID: <15F271AA-1F1A-4592-8B7E-005EAC67F7AF@unc.edu> > Content-Type: text/plain; charset=US-ASCII; delsp=yes; > format=flowed > > Hi, > > We had that happen once, and in the end the only thing that helped > was switching membrane manufacturers. The company swore they hadn't > changed anything, but neither had we, so. > > Maybe you can get free samples from a couple of different companies? > > Barbara MacGregor > > On May 2, 2008, at 1:06 PM, 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. salmon sperm DNA and Northern again (shiri shiri) > > > > From: shiri shiri > > Date: May 1, 2008 1:40:27 PM EDT > > To: > > Subject: salmon sperm DNA and Northern again > > > > > > Hello, > > > > You posted once on a problem you had with your northern hybridization. > > As you posted, I experience the same problem, unspecific binding > > all over > > the membrane, even in a blank one. I changed all my compounds, > > increased the > > concentration of the blocking solution but nothing works... > > The weird thing it worked great for many years but now no one in > > our lab, > > even ones who were previously successful in it, cannot solve this > > issue... > > > > I was wondering if you had a chance to solve this problem???? > > > > I would appreciate any help/suggestion > > > > THANKS > > SHIRI > > > > > > > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > ------------------------------ > > Message: 2 > Date: Fri, 2 May 2008 13:31:14 -0700 (PDT) > From: peter > Subject: EmulsionPCR > To: methods@net.bio.net > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hi group, I need to make emulsion in the emulsionPCR uniform. Any > suggestions? > Thanks, > Peter > > > ------------------------------ > > Message: 3 > Date: Fri, 2 May 2008 14:50:48 -0500 > From: "yan sun" > Subject: ethanol precipitation > To: Methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Dear all, > I did spin label DNA experiment. > I mixed my modified DNA with spin label material in DMF and fomamide > solution. I get 100% label efficiency. (the analytical gel shows that there > is only one spin label DNA band). > I was wondering how to purify my spin labeled DNA? My other site spin > labeled DNAs are not 100% spin label efficiency, so I RUN big PAGE gel, > and > use elutor to purified my target spin labeled DNA. during this process, I > lose quite amount material. that is why I don't want to run big gel this > time. > I made a research. looks like ethanol precipitation works. > anyone have experience, please feel free to share. > option 2. can I use freezing dry method? > > Thx a lot!!! > > > -- > Y. F.S. > > > ------------------------------ > > Message: 4 > Date: Sat, 3 May 2008 15:47:48 +0300 > From: Sarah > Subject: Cb5R Assay > To: methods@magpie.bio.indiana.edu > Message-ID: > > Content-Type: text/plain; charset=ISO-8859-1 > > Hello, > > I am looking for an assay to determine the activity of cytochrome b5 > reductase in blood. Can anyone help? > > Thank you. > > Sarah > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 36, Issue 3 > ************************************** > From gerchman from research.haifa.ac.il Sun May 11 14:10:29 2008 From: gerchman from research.haifa.ac.il (Yoram Gerchman) Date: Sun May 11 19:41:54 2008 Subject: Dyeing proteins BEFORE SDS-PAGE Message-ID: <1210533029.482744a5d7ed6@webmail.haifa.ac.il> Greetings netters Does anyone know of a suitable protocol for dyeing proteins BEFORE SDS-PAGE electrophoresis? I want to detect the proteins on a surface by dyeing, scrap them and run the gel. I can re-dye the gel with Coomassie in the end. Many thanks Yoram ------------------------------------------------------------------------ This message was sent using IMP, the Webmail Program of Haifa University From qau_mic from yahoo.com Mon May 12 05:16:40 2008 From: qau_mic from yahoo.com (sahar fatima) Date: Mon May 12 12:00:03 2008 Subject: SDS-PAGE Message-ID: <513782.3324.qm@web37304.mail.mud.yahoo.com> Hi, I need a protocol for SDS-PAGE. Also the composition, method of preparation of the gel and staining the proteins. Can acrude filtrat be run on the gel. Thanks --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. From qau_mic from yahoo.com Mon May 12 05:20:45 2008 From: qau_mic from yahoo.com (sahar fatima) Date: Mon May 12 12:00:08 2008 Subject: Actinomycetes identification Message-ID: <344907.8654.qm@web37306.mail.mud.yahoo.com> Hi. The primers used for bacteria for moleculer identification by amplification of 16s rRNA can be used for actinomycetes or not? If not then please tell which primers to be used for their identification. Thanks --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. From vadivelarunachalam from yahoo.com Mon May 12 03:04:52 2008 From: vadivelarunachalam from yahoo.com (V Arunachalam) Date: Mon May 12 12:00:14 2008 Subject: transcription factor assay Zinc finger homeodomain Message-ID: <409656.90074.qm@web94707.mail.in2.yahoo.com> Dear All I am interested in analysing a clone from a legume plant which matches Arabdiposis Zinc finger homoedomain. Could anyone help me in?locating the?sources of commercial antibodies for this protein domain. ?With best wishes, V.Arunachalam Sr Scientist Horticulture Genetics lab. CPCRI Kasaragod 671 124 Kerala DBT Postdoctoral Fellow Genetic transformation Lab. ICRISAT Patancheru 502324 AP India E-mail : vadivelarunachalam@yahoo.com From Chandigarh to Chennai - find friends all over India. Go to http://in.promos.yahoo.com/groups/citygroups/ From z_hattab from yahoo.com Sun May 11 14:16:59 2008 From: z_hattab from yahoo.com (dana hattab) Date: Mon May 12 12:00:26 2008 Subject: fragment analysi Message-ID: <479358.2317.qm@web58006.mail.re3.yahoo.com> hi how can i overcome the non-specific amplification in gene scan? and is the stutter artifact normal or it will interfere with my data analysis? Dana Hattab --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. From bmacgreg from unc.edu Mon May 12 12:25:40 2008 From: bmacgreg from unc.edu (Barbara MacGregor) Date: Mon May 12 23:05:54 2008 Subject: Actinomyces identification In-Reply-To: <200805121703.m4CH3SO09562@net.bio.net> References: <200805121703.m4CH3SO09562@net.bio.net> Message-ID: <6331AE1C-EBB7-4186-B9CA-E051A2793437@unc.edu> > > From: sahar fatima > Date: May 12, 2008 6:20:45 AM EDT > To: methods@magpie.bio.indiana.edu > Subject: Actinomycetes identification > > > Hi. > The primers used for bacteria for moleculer identification by > amplification of 16s rRNA can be used for actinomycetes or not? If > not then please tell which primers to be used for their > identification. > Thanks > Hello, It depends which primers you are talking about... The ProbeBase site (www.microbial-ecology.de/probebase/) is one convenient place you can check. Barbara MacGregor From R.Jayakumar from roswellpark.org Mon May 12 16:14:24 2008 From: R.Jayakumar from roswellpark.org (Jayakumar, R) Date: Mon May 12 23:06:07 2008 Subject: SDS-PAGE In-Reply-To: <513782.3324.qm@web37304.mail.mud.yahoo.com> References: <513782.3324.qm@web37304.mail.mud.yahoo.com> Message-ID: <97101976F8A044468CA74FE11883B90E173E7A62@VISTA.roswellpark.org> Check "Molecular cloning manual" (sambrook) or the "Current protocols in Molecular biology" (the red book). These are very basic techniques and should and must be available in biochemistry or molecular biology lab. Jay -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of sahar fatima Sent: Monday, May 12, 2008 6:17 AM To: methods@magpie.bio.indiana.edu Subject: SDS-PAGE Hi, I need a protocol for SDS-PAGE. Also the composition, method of preparation of the gel and staining the proteins. Can acrude filtrat be run on the gel. Thanks --------------------------------- Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it 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 scrampto from uci.edu Mon May 12 14:41:28 2008 From: scrampto from uci.edu (Steve Crampton) Date: Mon May 12 23:06:13 2008 Subject: Luciferase Assay! Message-ID: <96ACDEE6-9621-4934-AA7B-657CAD2B598D@uci.edu> For luciferase assays, we always use the one-to-one proportion (100uL of lysate:100uL of substrate). Looks like you were on the right track! We also assay cells from 6-well plates and Steve Message: 2 Date: Thu, 8 May 2008 20:01:10 +0530 From: "Sudheendra Rao N R" Subject: Luciferase Assay! To: methods , methods@magpie.bio.indiana.edu Message-ID: Content-Type: text/plain; charset=ISO-8859-1 Hi all, 1. I am doing Luciferase assay to understand transcriptional regulation. I was looking for proper reporter lysis buffers. i found out one with KH2PO4 (7.8 pH) with 0.2% Triton X-100 (v/v)..is there any better? it takes time for us to order promega 5x reporter lysis buffer. 2. i am doing duplicate assays in 6 well plates..and collecting cells in pbs and lysing them in 200uL buffer. however i am not sure what is the "standard" lysate:luciferase substrate (roche) proportion. i have tried 10uL:90uL, 40uL:60uL, 100:100uL combinations, later gives me better RLUs we use a stand-alone, tube-luminometer (sirius), and do not use injectors. please let me know. (i will post my questions on DLuciferase Assay also..soon :)) thanks in advance sudheendra. From sioklian from gmail.com Tue May 13 06:50:17 2008 From: sioklian from gmail.com (lsl) Date: Tue May 13 09:02:27 2008 Subject: DNA Solution - Some Queries Message-ID: <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> Hello. I hope you can help me to solve some of the problems below. 1) We purchase the synthetic oligonucleotides and they claim to have 100uM of DNA. Is this number reliable for each and every batch? 2) If what is claimed by the oligo supplier is not really correct, usually what is the method you used to prepare certain concentration of DNA solution (say 10uM)? 3) How to use UV-vis to check the concentration of a DNA solution and how to calculate the amount of DNA present in the solution? If possible, any books or references that you can recommend? Thank you very much for you time. lsl From blackhole from abuse.plus.com Tue May 13 07:29:49 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Tue May 13 09:02:35 2008 Subject: DNA Solution - Some Queries References: <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> Message-ID: Historians believe that in newspost <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> on Tue, 13 May 2008, lsl penned the following literary masterpiece: >1) We purchase the synthetic oligonucleotides and they claim to have >100uM of DNA. Is this number reliable for each and every batch? Yield varies batch to batch, oligo to oligo. >2) If what is claimed by the oligo supplier is not really correct, >usually what is the method you used to prepare certain concentration >of DNA solution (say 10uM)? Go to an oligo calculator site say IDT or any primer design program. http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/ Feed in your oligo sequence and it will come back with a figure of nmol/OD and ug/OD i.e. 1 OD260 of a 20mer I juts checked here is calculated as 4.82nmol or 30.2ug. >3) How to use UV-vis to check the concentration of a DNA solution and >how to calculate the amount of DNA present in the solution? If >possible, any books or references that you can recommend? Start with a Quartz cuvette. I resuspend my 40nmol oligo stocks in sterile 300ul water or TE (10mM Tris pH 8.0, 0.1mM EDTA). Measure 2ul in 1ml water at OD260. From OD calculate total no. of OD's in 300ul and then from the calculator figures, stock concn. in pM/ul. I would then dilute some of the stock to 10 or 25pM/ul for use in PCR. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From office from rvs-ruse.com Tue May 13 01:21:04 2008 From: office from rvs-ruse.com (rvs-ruse.com) Date: Tue May 13 09:13:33 2008 Subject: Actinobacillu pleuropneumoniae antiserum Message-ID: <001701c8b4c1$865822f0$0201a8c0@hoffice> Dear colleagues, We would like to produce polyclonal antibodies against Actinobavillus pleuropneumonia. I am looking for good detailed protocol! Thank you in advance! Ivaylo Nankov Naydenov, DVM, MSc, CEO Regional Veterinary Station (RVS - Ruse EOOD) 3 Maritza str. Ruse 7003 Bulgaria ICQ 35690743 skype# ivaynanay office@rvs-ruse.com ivaynanay@gmail.com From josenet from tiscali.co.uk Tue May 13 08:53:50 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Tue May 13 16:54:59 2008 Subject: DNA Solution - Some Queries References: <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> Message-ID: <68tl2uF2ueht7U1@mid.individual.net> "lsl" wrote in message news:7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com... > Hello. I hope you can help me to solve some of the problems below. > 1) We purchase the synthetic oligonucleotides and they claim to have > 100uM of DNA. Is this number reliable for each and every batch? in addition to what Duncan said, don't confuse 100uM (a concentration) with 100umoles (an amount), as the question above seems to imply. I see this too often in the lab. Jose From hroychow from nmsu.edu Tue May 13 13:24:26 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Tue May 13 16:55:08 2008 Subject: DNA Solution - Some Queries In-Reply-To: <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> References: <7e3e00a7-f5c2-4636-bad7-98263e35f13a@t54g2000hsg.googlegroups.com> Message-ID: <2659.128.123.174.0.1210703066.squirrel@webmail.nmsu.edu> Hmmm .... In my experience, the molarity is fairly accurate, and certainly is not generally afactor for routine use of the oligos. Now, if one has to ask the next two questions, wouldn't it be better that the downstream apllications are also left up to some commercial outfit? Should any in the lab have to ask these questions, such a lab is in a rather bad shape! > Hello. I hope you can help me to solve some of the problems below. > 1) We purchase the synthetic oligonucleotides and they claim to have > 100uM of DNA. Is this number reliable for each and every batch? > 2) If what is claimed by the oligo supplier is not really correct, > usually what is the method you used to prepare certain concentration > of DNA solution (say 10uM)? > 3) How to use UV-vis to check the concentration of a DNA solution and > how to calculate the amount of DNA present in the solution? If > possible, any books or references that you can recommend? > Thank you very much for you time. > > lsl > _______________________________________________ > 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 office from rvs-ruse.com Tue May 13 10:20:33 2008 From: office from rvs-ruse.com (rvs-ruse.com) Date: Wed May 14 11:27:02 2008 Subject: APP antiserum Message-ID: <000f01c8b50c$e37cefb0$0201a8c0@hoffice> Dear colleagues, We would like to produce polyclonal antibodies against Actinobavillus pleuropneumonia. I am looking for good detailed protocol! Thank you in advance! Ivaylo Nankov Naydenov, DVM, MSc, CEO Regional Veterinary Station (RVS - Ruse EOOD) 3 Maritza str. Ruse 7003 Bulgaria ICQ 35690743 skype# ivaynanay office@rvs-ruse.com ivaynanay@gmail.com From metugenetics from yahoo.com Wed May 14 16:26:27 2008 From: metugenetics from yahoo.com (Ozan Aygun) Date: Wed May 14 17:47:51 2008 Subject: Blunt End Concatemerization Message-ID: <831655.71464.qm@web90407.mail.mud.yahoo.com> Dear all, I have annealed two complementary 20nt oligos to give a blunt end fragment and would like to concatemerize it to give a tandem copies of >15 fragments. I have tried Roche T4 ligase without any success with standard buffer conditions in a 50ul volume. Do you have any alternative suggestions? Many thanks, Ozan From debashish.das from ki.se Wed May 14 13:00:35 2008 From: debashish.das from ki.se (Debashish Das) Date: Wed May 14 17:47:58 2008 Subject: CDK6 kinase assay Message-ID: Dear all I would need your help and all suggestions for doing a kinase assay in-vitro to detect the activity of CDK6. I have never done a kinase assay and as I see the papers published, they have all used only radioactive detection method!!. It would be of great help if anyone around has had experience in kinase assay preferably in looking for the CDK6 or similar kinase activities. all help and suggestions are highly appreciated and I would be deeply grateful for them a protocol and also if possible a source from where to get the reagents can help a lot more.. thanking you all for your time and help best of things deb From josenet from tiscali.co.uk Wed May 14 20:23:41 2008 From: josenet from tiscali.co.uk (Jose de las Heras) Date: Wed May 14 22:05:18 2008 Subject: Blunt End Concatemerization References: Message-ID: <691hl9F2u4taeU1@mid.individual.net> "Ozan Aygun" wrote in message news:mailman.90.1210805268.3533.methods@net.bio.net... > Dear all, > > I have annealed two complementary 20nt oligos to give > a blunt end fragment and would like to concatemerize > it to give a tandem copies of >15 fragments. I have > tried Roche T4 ligase without any success with > standard buffer conditions in a 50ul volume. > > Do you have any alternative suggestions? > > Many thanks, > > Ozan Are the oligos phosphorylated? usually you will get dephosphorylated oligos, unless you ask specifically, and these won't ligate. Jose From peter.ianakiev from gmail.com Wed May 14 20:39:20 2008 From: peter.ianakiev from gmail.com (peter) Date: Wed May 14 22:05:23 2008 Subject: APP antiserum References: Message-ID: On May 13, 11:20 am, "rvs-ruse.com" wrote: > Dear colleagues, > We would like to produce polyclonal antibodies against Actinobavillus pleuropneumonia. I am looking for good detailed protocol! > > Thank you in advance! > > Ivaylo Nankov Naydenov, DVM, MSc, CEO > Regional Veterinary Station (RVS - Ruse EOOD) > 3 Maritza str. > Ruse 7003 > Bulgaria > ICQ 35690743 > skype# ivaynanay > off...@rvs-ruse.com > ivayna...@gmail.com Dear Ivaylo, You don't have to "produce" - just get recently infected pig and isolate Ab or serum from it.... From sudhee26 from gmail.com Wed May 14 23:44:02 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 15 10:50:18 2008 Subject: CDK6 kinase assay In-Reply-To: References: Message-ID: Hi, check this out *In Vitro Kinase Assay (**33* *).* NRK (5 ? 106) and 5 ? 105 K6D1 cells were lysed with 2 ml and 0.5 ml of ice-cold immunoprecipitation buffer consisting of 50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 21 mM DTT, 0.1% Tween-20, 10% glycerol, 1 mM PMSF, 4 mg/ml each of leupeptin, pepstatin, and aprotinin, 0.1 M NaF, 2 mM sodium orthovanadate, and 10 mM [image: beta]-glycerophosphate, respectively. Lysates were incubated at 4?C for 2 hr with 0.3 ?g of [image: alpha]Cdk4, [image: alpha]Cdk6, or [image: alpha]Cdk2. Protein G-Sepharose suspension (15 ?l) (Amersham-Pharmacia) then was added and incubated at 4?C for an additional 1 hr. Immune complexes bound to Protein G-Sepharose were precipitated by centrifugation and washed with ice-cold, glycerol-free immunoprecipitation buffer. The immunopurified Cdk4, Cdk6, and Cdk2 were incubated at 30?C for 30 min in 10 ?l of reaction buffer (50 mM Hepes, pH 7.5/1 mM EGTA/10 mM KCl/10 mM MgCl2/1 mM DTT) containing 5 ?g of truncated Rb (QED Bioscience, San Diego) and 10 mM ATP. The reaction products were electrophoresed on 10% SDS-polyacrylamide gels and transferred to poly(vinylidene difluoride) membrane filters. The Rb molecules phosphorylated by Cdk4 or Cdk6 were immunodetected with the anti-Ser-780-phosphorylated Rb antibody (MBL), and the Rb phosphorylated by Cdk2 was immunodetected with the anti-Ser811-phosphorylated Rb antibody (New England Biolabs). http://www.pnas.org/cgi/content/full/96/23/13197?ck=nck#F5 Sudheendra. On Wed, May 14, 2008 at 11:30 PM, Debashish Das wrote: > Dear all > > I would need your help and all suggestions for doing a kinase assay > in-vitro to detect the > activity of CDK6. I have never done a kinase assay and as I see the papers > published, they > have all used only radioactive detection method!!. It would be of great > help if anyone around > has had experience in kinase assay preferably in looking for the CDK6 or > similar kinase > activities. > > all help and suggestions are highly appreciated and I would be deeply > grateful for them > > a protocol and also if possible a source from where to get the reagents can > help a lot more.. > > thanking you all for your time and help > > best of things > > deb > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From rmezencev3 from gatech.edu Thu May 15 10:04:03 2008 From: rmezencev3 from gatech.edu (Mezencev, Roman) Date: Thu May 15 10:50:43 2008 Subject: DNA Solution - Some Queries In-Reply-To: <463992582.2740811210863725504.JavaMail.root@mail6.gatech.edu> Message-ID: <2116157837.2741401210863843639.JavaMail.root@mail6.gatech.edu> You can use UV spectrophotometry for determination of ssDNA concentration as follows: at 260 nm, OD value of 1 corresponds to about 40 ug/mL of single-stranded DNA. So from OD at 260 and molecular weight of your oligos you can calculate concentration of your ssDNA in uM: c = OD x 40,000 / M, where M is molecular weight of your oligo in g/mol. You can find many suitable references for this procedure. RM From debashish.das from ki.se Thu May 15 07:34:35 2008 From: debashish.das from ki.se (Debashish Das) Date: Thu May 15 10:50:49 2008 Subject: Cyclin D1 promoter Message-ID: Hi I am interested in looking for the promoter of Cyclin D1 and compare that across species. I would be very grateful if anyone here could provide me with any website where not only can I find the promoter of the gene of my interest as well as would be able to compare it across the species. thanking you all in advance! From maximilianh from gmail.com Thu May 15 11:25:34 2008 From: maximilianh from gmail.com (Maximilian Haeussler) Date: Thu May 15 12:57:52 2008 Subject: Cyclin D1 promoter In-Reply-To: References: Message-ID: <76f031ae0805150925rbc90581yc0ede535fda52bcd@mail.gmail.com> I think this question should be sent to a bioinformatics-mailing-list like the BBB (http://www.bioinformatics.org/pipermail/bio_bulletin_board/) Short answer: Depends on what do you want to find. I guess you want to compare binding sites. I assume that you don't want to align them, (if you did, then simply type dna alignment into google) Links of all of the tools that I am citing here can be found at the Wiki for tools on Transcriptional Regulation on openwetware.com: http://openwetware.org/wiki/Tregwiki:Main_Page I dont' think that there is a very usable tool for this task. Comparison of un-alignable cis-regulatory sequences is very strange topic and there is not good usable tool yet. You should have an idea of the type of transcription factor that is binding there. But you can try Matinspector from genomatix in any case, it's very easy to use. There is FamilyRelations from Titus Brown which can compare short words in a set of sequences and is creating a graphical representation of them. There are some recent publications that have been benchmarked on the even-skipped enhancer in drosophila and they can often find the orthologous enhancer in other un-alignable species but they are usually not available as websites and you better know a bit about programming to use them. cheers, Max 2008/5/15 Debashish Das : > Hi > > I am interested in looking for the promoter of Cyclin D1 and compare that > across species. I > would be very grateful if anyone here could provide me with any website > where not only can > I find the promoter of the gene of my interest as well as would be able to > compare it across > the species. > > thanking you all in advance! > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Maximilian Haeussler, tel: +33 6 12 82 76 16 From ebs15242 from creighton.edu Thu May 15 13:49:59 2008 From: ebs15242 from creighton.edu (Ed Siefker) Date: Thu May 15 16:54:52 2008 Subject: Cyclin D1 promoter In-Reply-To: References: Message-ID: <482C85D7.1000806@creighton.edu> dcode.org has a number of comparative genomics tools that you might find helpful. Debashish Das wrote: > Hi > > I am interested in looking for the promoter of Cyclin D1 and compare that across species. I > would be very grateful if anyone here could provide me with any website where not only can > I find the promoter of the gene of my interest as well as would be able to compare it across > the species. > > thanking you all in advance! > > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods From qau_mic from yahoo.com Fri May 16 09:02:21 2008 From: qau_mic from yahoo.com (sahar fatima) Date: Fri May 16 11:13:58 2008 Subject: Identification of Actinomycetes Message-ID: <900108.58555.qm@web37303.mail.mud.yahoo.com> Hi, Can anyone tell me a Pcr primer for identification of actinomycetes by amplifying 16S ribosomal RNA region of the genomic DNA by PCR. From yvonne.couch from dpag.ox.ac.uk Fri May 16 11:56:30 2008 From: yvonne.couch from dpag.ox.ac.uk (Yvonne Couch) Date: Fri May 16 20:25:21 2008 Subject: Standard Curve vs DDCT Message-ID: <013301c8b775$ca10bb90$2e7a01a3@dpag.ox.ac.uk> Hi guys, I've recently had some problems getting RNA for some qPCR but my main problem is that I run a standard curve with all my sets of primers to make sure I can compare the efficiency of the reaction properly. With my current RNA yield I don't have enough to run a standard curve. There are differences of opinion in our lab, one person says always run a standard curve to make sure the efficiency is the comparable and one person says you don't need a standard curve just use the controls to compare levels and do Delta:Delta CT. Any thoughts? Cheers Yvonne From higginsb78 from gmail.com Fri May 16 21:26:56 2008 From: higginsb78 from gmail.com (Brian P Higgins) Date: Sat May 17 13:34:47 2008 Subject: Standard Curve vs DDCT In-Reply-To: <013301c8b775$ca10bb90$2e7a01a3@dpag.ox.ac.uk> References: <013301c8b775$ca10bb90$2e7a01a3@dpag.ox.ac.uk> Message-ID: What are you running your standard with? I would just clone your PCR products, then use the miniprep to run your standard curve--you'll never run out of standard. Some are fans of of the ddCt method. There are some very detailed versions of the ddCt method (Pfall comes to mind, along with another version called REST), but I simply don't trust it. There's a recent review by Bustin (within the past couple years) that addresses the issue of quantitating RNA via qPCR. The basic problem with the ddCt method is that it assumes equal efficiencies among all your primer pairs. There may be ways to take this into account now, but I have always treated each primer pair separately, then done the math after the fact. Regardless of the method you choose for quantitation, I would run a standard curve for each primer pair--I think Bustin points out in his review that a small difference in PCR efficiency can lead to gross miscalculations. To run the standards, just clone your PCR products and run serial dilutions of the plasmid. good luck brian On Fri, May 16, 2008 at 12:56 PM, Yvonne Couch wrote: > Hi guys, > > I've recently had some problems getting RNA for some qPCR but my main > problem is that I run a standard curve with all my sets of primers to make > sure I can compare the efficiency of the reaction properly. With my > current > RNA yield I don't have enough to run a standard curve. There are > differences of opinion in our lab, one person says always run a standard > curve to make sure the efficiency is the comparable and one person says you > don't need a standard curve just use the controls to compare levels and do > Delta:Delta CT. > > Any thoughts? > > Cheers > > Yvonne > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From ben.long from yourfinger.anu.edu.au Sun May 18 20:24:40 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Sun May 18 22:09:33 2008 Subject: IP Protocols and hints Message-ID: <4830d6d8$1@clarion.carno.net.au> Hi all, I've recently got my hands on an antibody to a small protein that's part of an enormous complex (over 100 nm in diameter) and thought I might try some IP to purify it. I've also got some Protein G-sepharose beads in the fridge which I could use for IP. Can anyone offer protocols or point me in the direction of some to help me out? I'll need to do this under native conditions. I've done very little IP before, so any hints would be handy. Cheers, Bean From sonali_mookerjee from yahoo.com Mon May 19 15:26:35 2008 From: sonali_mookerjee from yahoo.com (SONALI MOOKERJEE) Date: Mon May 19 20:24:44 2008 Subject: In situ hybridization with DNA probe Message-ID: <857465.97700.qm@web53601.mail.re2.yahoo.com> I am planning to use in situ hybridization to detect gene expression patterns in plant tissue. Most of the protocols I have found use RNA probes although they acknowledge that using RNA probes is more difficult. Does anyone have a protocol for using DNA probes with DIG labelling? Or can anybody point to any publication that describes the technique? Thanks Sonali Mookerjee Michigan State University From jyyoungjimmy from gmail.com Mon May 19 20:38:22 2008 From: jyyoungjimmy from gmail.com (Jim Young) Date: Mon May 19 23:03:15 2008 Subject: Methods Digest, Vol 36, Issue 16 In-Reply-To: <200805191704.m4JH4AO02119@net.bio.net> References: <200805191704.m4JH4AO02119@net.bio.net> Message-ID: <57f211620805191838m32fcbf20h8099e243acccb86e@mail.gmail.com> Hi, If you want to go further for IP-Western, a product will be very helpful to you. The kit is optimized to heavy chains, light chains contamination. What more excited! The kit cut the Western time to one hour. See here: http://www.genscript.com/ip_western_tech.html On Tue, May 20, 2008 at 1:04 AM, 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. IP Protocols and hints (Bean Long) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Mon, 19 May 2008 11:24:40 +1000 > From: Bean Long > Subject: IP Protocols and hints > To: methods@net.bio.net > Message-ID: <4830d6d8$1@clarion.carno.net.au> > Content-Type: text/plain; charset=ISO-8859-1; format=flowed > > Hi all, > > I've recently got my hands on an antibody to a small protein that's part > of an enormous complex (over 100 nm in diameter) and thought I might try > some IP to purify it. I've also got some Protein G-sepharose beads in > the fridge which I could use for IP. Can anyone offer protocols or point > me in the direction of some to help me out? I'll need to do this under > native conditions. I've done very little IP before, so any hints would > be handy. > > Cheers, > > Bean > > > ------------------------------ > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 36, Issue 16 > *************************************** > From agbiok4 from gmail.com Mon May 19 22:29:56 2008 From: agbiok4 from gmail.com (kamalaker nasani) Date: Mon May 19 23:03:24 2008 Subject: DNA EXTRACTION Message-ID: I am working wth okra(Bhendi). As we all know that bhendi will be having lot of mucelage. It became a big problem for DNA extration. And llso for protein extraction for ELISA. Please if anybody have any information related to this please share with me. Thanx in advance -- URS Kamalaker Nasani "The illiterate of the 21st Century will not be those who cannot read or write. The illiterate will be those who cannot learn, unlearn and relearn" -George Bernard Shaw From M.Dunowska from massey.ac.nz Tue May 20 19:07:36 2008 From: M.Dunowska from massey.ac.nz (Dunowska, Magda) Date: Wed May 21 21:24:04 2008 Subject: DNA EXTRACTION In-Reply-To: References: Message-ID: <92FDFD8B26EB6542B1E1BF017BB998D1480CC865C2@TUR-EXCHMBX.massey.ac.nz> Hi, A friend of mince was working with mollusc tissues, and had a similar problem. He had to abandon any DNA extraction kits and went back to phenol-chloroform extraction and ethanol precipitation. As far as I know, this worked for him. Maybe you could try trizol if you also want proteins? Magda Magda Dunowska, LW, PhD Senior Lecturer in Veterinary Infectious Diseases (Virology) Institute of Veterinary, Animal and Biomedical Sciences Te Kura Mātauranga Kararehe Massey University Palmerston North New Zealand Phone : (06) 350-5799 ext 7571 Website : http://ivabs.massey.ac.nz -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of kamalaker nasani Sent: Tuesday, 20 May 2008 3:30 p.m. To: methods@magpie.bio.indiana.edu Subject: DNA EXTRACTION I am working wth okra(Bhendi). As we all know that bhendi will be having lot of mucelage. It became a big problem for DNA extration. And llso for protein extraction for ELISA. Please if anybody have any information related to this please share with me. Thanx in advance -- URS Kamalaker Nasani "The illiterate of the 21st Century will not be those who cannot read or write. The illiterate will be those who cannot learn, unlearn and relearn" -George Bernard Shaw _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods From hroychow from nmsu.edu Tue May 20 20:50:31 2008 From: hroychow from nmsu.edu (Dr. Hiranya S. Roychowdhury) Date: Wed May 21 21:24:11 2008 Subject: DNA EXTRACTION In-Reply-To: References: Message-ID: <1784.71.210.220.185.1211334631.squirrel@webmail.nmsu.edu> It would be difficult to suggest anything without knowing what you have tried already. > I am working wth okra(Bhendi). As we all know that bhendi will be having > lot of mucelage. It became a big problem for DNA extration. And llso for > protein extraction for ELISA. Please if anybody have any information > related to this please share with me. > > > Thanx in advance > > -- > URS > Kamalaker Nasani > > > > "The illiterate of the 21st Century will not be those > who cannot read or write. The illiterate will be those > who cannot learn, unlearn and relearn" > -George Bernard Shaw > _______________________________________________ > 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 blackhole from abuse.plus.com Wed May 21 10:12:17 2008 From: blackhole from abuse.plus.com (Duncan Clark) Date: Wed May 21 21:24:35 2008 Subject: Standard Curve vs DDCT References: <013301c8b775$ca10bb90$2e7a01a3@dpag.ox.ac.uk> Message-ID: Historians believe that in newspost on Fri, 16 May 2008, Brian P Higgins penned the following literary masterpiece: >To >run the standards, just clone your PCR products and run serial dilutions of >the plasmid. And use linearised plasmid, not supercoiled plasmid. Duncan -- I love deadlines. I especially like the whooshing noise they make as they go flying by. Duncan Clark GeneSys Ltd. From engelbert_buxbaum from hotmail.com Wed May 21 10:28:17 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed May 21 21:24:46 2008 Subject: Dyeing proteins BEFORE SDS-PAGE References: Message-ID: Am 11.05.2008, 15:10 Uhr, schrieb Yoram Gerchman : > > Greetings netters > Does anyone know of a suitable protocol for dyeing proteins BEFORE > SDS-PAGE > electrophoresis? I want to detect the proteins on a surface by dyeing, > scrap > them and run the gel. I can re-dye the gel with Coomassie in the end. > Many thanks > Yoram That should be possible with non-covalently binding dyes like amidoblack, coomassie or Ponceau, provided the surface does not bind them. You may have to play a little with the dilution. From engelbert_buxbaum from hotmail.com Wed May 21 10:31:05 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed May 21 21:24:50 2008 Subject: SDS-PAGE References: Message-ID: Am 12.05.2008, 06:16 Uhr, schrieb sahar fatima : > Hi, > I need a protocol for SDS-PAGE. Also the composition, method of > preparation of the gel and staining the proteins. Can acrude filtrat be > run on the gel. @article{Lae-70, AUTHOR= {U.K. Laemli}, TITLE= {Cleavage of structural proteins during the assembly of the head of bacteriophage {T}4}, YEAR= {1970}, JOURNAL= {Nature}, PAGES= {680-685}, VOLUME= {227}, LANGUAGE= {engl} } @book{Cra-85, AUTHOR= {A. Chrambach}, TITLE= {The practice of quantitative gel electrophoresis}, YEAR= {1985}, PUBLISHER= {VCH}, ADDRESS= {Weinheim}, LANGUAGE= {engl} } From engelbert_buxbaum from hotmail.com Wed May 21 10:46:45 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Wed May 21 21:24:55 2008 Subject: APP antiserum References: Message-ID: Am 13.05.2008, 11:20 Uhr, schrieb rvs-ruse.com : > Dear colleagues, > We would like to produce polyclonal antibodies against Actinobavillus > pleuropneumonia. I am looking for good detailed protocol! @book{Har-88, AUTHOR= {E. Harlow and D. Lane}, TITLE= {Antibodies --- A Laboratory Manual}, YEAR= {1988}, PUBLISHER= {Cold Spring Harbor Laboratory Press}, ADDRESS= {Cold Spring Harbor}, EDITION= {1}, ISBN= {978-0879694145}, LANGUAGE= {engl} } From sudhee26 from gmail.com Wed May 21 21:17:01 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed May 21 21:25:00 2008 Subject: DNA EXTRACTION In-Reply-To: References: Message-ID: Hi, go through the following PDF. It also gives references which might be useful. I would suggest try "Roots" also, after washing them and disinfecting them.. http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb18/R00-056.pdf all the best. Sudheendra. On Tue, May 20, 2008 at 8:59 AM, kamalaker nasani wrote: > I am working wth okra(Bhendi). As we all know that bhendi will be having > lot of mucelage. It became a big problem for DNA extration. And llso for > protein extraction for ELISA. Please if anybody have any information > related to this please share with me. > > > Thanx in advance > > -- > URS > Kamalaker Nasani > > > > "The illiterate of the 21st Century will not be those > who cannot read or write. The illiterate will be those > who cannot learn, unlearn and relearn" > -George Bernard Shaw > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Wed May 21 21:17:01 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Wed May 21 21:25:05 2008 Subject: DNA EXTRACTION In-Reply-To: References: Message-ID: Hi, go through the following PDF. It also gives references which might be useful. I would suggest try "Roots" also, after washing them and disinfecting them.. http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb18/R00-056.pdf all the best. Sudheendra. On Tue, May 20, 2008 at 8:59 AM, kamalaker nasani wrote: > I am working wth okra(Bhendi). As we all know that bhendi will be having > lot of mucelage. It became a big problem for DNA extration. And llso for > protein extraction for ELISA. Please if anybody have any information > related to this please share with me. > > > Thanx in advance > > -- > URS > Kamalaker Nasani > > > > "The illiterate of the 21st Century will not be those > who cannot read or write. The illiterate will be those > who cannot learn, unlearn and relearn" > -George Bernard Shaw > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Wed May 21 21:49:26 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 22 10:00:07 2008 Subject: Dyeing proteins BEFORE SDS-PAGE In-Reply-To: References: Message-ID: any help?? http://www.bio.net/bionet/mm/proteins/1994-March/001053.html Sudheendra. On Wed, May 21, 2008 at 8:58 PM, Dr Engelbert Buxbaum < engelbert_buxbaum@hotmail.com> wrote: > Am 11.05.2008, 15:10 Uhr, schrieb Yoram Gerchman < > gerchman@research.haifa.ac.il>: > > >> Greetings netters >> Does anyone know of a suitable protocol for dyeing proteins BEFORE >> SDS-PAGE >> electrophoresis? I want to detect the proteins on a surface by dyeing, >> scrap >> them and run the gel. I can re-dye the gel with Coomassie in the end. >> Many thanks >> Yoram >> > > That should be possible with non-covalently binding dyes like amidoblack, > coomassie or Ponceau, provided the surface does not bind them. You may have > to play a little with the dilution. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Wed May 21 21:49:26 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 22 10:00:28 2008 Subject: Dyeing proteins BEFORE SDS-PAGE In-Reply-To: References: Message-ID: any help?? http://www.bio.net/bionet/mm/proteins/1994-March/001053.html Sudheendra. On Wed, May 21, 2008 at 8:58 PM, Dr Engelbert Buxbaum < engelbert_buxbaum@hotmail.com> wrote: > Am 11.05.2008, 15:10 Uhr, schrieb Yoram Gerchman < > gerchman@research.haifa.ac.il>: > > >> Greetings netters >> Does anyone know of a suitable protocol for dyeing proteins BEFORE >> SDS-PAGE >> electrophoresis? I want to detect the proteins on a surface by dyeing, >> scrap >> them and run the gel. I can re-dye the gel with Coomassie in the end. >> Many thanks >> Yoram >> > > That should be possible with non-covalently binding dyes like amidoblack, > coomassie or Ponceau, provided the surface does not bind them. You may have > to play a little with the dilution. > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From kgilbride from shire.com Thu May 22 08:22:02 2008 From: kgilbride from shire.com (Gilbride, Kevin) Date: Thu May 22 10:00:41 2008 Subject: SDS-PAGE In-Reply-To: Message-ID: <0F2CFCE6FC067A42813DFA1F9BB0697C01214AF4@SHCHBEX03.corp.shire.com> Biorad's catalog used to have all this info or you can try Hoefer's site -----Original Message----- From: methods-bounces@oat.bio.indiana.edu [mailto:methods-bounces@oat.bio.indiana.edu] On Behalf Of Dr Engelbert Buxbaum Sent: Wednesday, May 21, 2008 11:31 AM To: methods@magpie.bio.indiana.edu Subject: Re: SDS-PAGE Am 12.05.2008, 06:16 Uhr, schrieb sahar fatima : > Hi, > I need a protocol for SDS-PAGE. Also the composition, method of > preparation of the gel and staining the proteins. Can acrude filtrat be > run on the gel. @article{Lae-70, AUTHOR= {U.K. Laemli}, TITLE= {Cleavage of structural proteins during the assembly of the head of bacteriophage {T}4}, YEAR= {1970}, JOURNAL= {Nature}, PAGES= {680-685}, VOLUME= {227}, LANGUAGE= {engl} } @book{Cra-85, AUTHOR= {A. Chrambach}, TITLE= {The practice of quantitative gel electrophoresis}, YEAR= {1985}, PUBLISHER= {VCH}, ADDRESS= {Weinheim}, LANGUAGE= {engl} } _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ______________________________________________________________ This message has been scanned for all viruses by BTnet VirusScreen. The service is delivered in partnership with MessageLabs. This service does not scan any password protected or encrypted attachments. 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If received in error, please delete this email and any attachments and confirm this to the sender. www.shire.com From e_barouki from hotmail.com Thu May 22 04:17:07 2008 From: e_barouki from hotmail.com (Emad) Date: Thu May 22 10:00:47 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: <200805201704.m4KH48O11148@net.bio.net> Message-ID: Hello, I am doing real time PCR for analysing my genes expression under different condition of my fungus. We use SYPR green. Actually I isolated RNA using both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on column following the kit's instruction. Unfortunately I could still detect a DNA contamination!! I treated the RNA again with DNase from NEB but still some traces of DNA exist. I tried to avoid the DNase treatment because I learned from some people who deal with RT-PCR to avoid DNase treatment and used Intron Span primers, and here is the big suffering, I used 18-20mere primers which locate just in the middle of exon borders and still these primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and still get some amplification, isn't that terrible? I thought to go to 3 bases at the 3' end of the primer but then you don't get any good prime which suit in this area!!? Can somebody please give me some hints or has any experience in such a good DNase which doesn't leave any DNA traces?! I am not optimistic in that. Emad From sudhee26 from gmail.com Thu May 22 11:47:55 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 22 12:33:15 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: References: <200805201704.m4KH48O11148@net.bio.net> Message-ID: Hey, i have never isolated RNA using columns..but just Trizol and rarely i get DNA contamination. It is surprising that DNAse still not able to digest your DNA i suggest u to run following control lanes in the gel 1. just DNA loading Dye 2. control DNA incubated with DNAse 3. Just DNAse i guess that must trouble shoot it. Sudheendra. On Thu, May 22, 2008 at 2:47 PM, Emad wrote: > Hello, > > I am doing real time PCR for analysing my genes expression under different > condition of my fungus. We use SYPR green. Actually I isolated RNA using > both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on > column following the kit's instruction. Unfortunately I could still detect > a > DNA contamination!! I treated the RNA again with DNase from NEB but still > some traces of DNA exist. I tried to avoid the DNase treatment because I > learned from some people who deal with RT-PCR to avoid DNase treatment and > used Intron Span primers, and here is the big suffering, I used 18-20mere > primers which locate just in the middle of exon borders and still these > primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and > still > get some amplification, isn't that terrible? I thought to go to 3 bases at > the 3' end of the primer but then you don't get any good prime which suit > in > this area!!? > > Can somebody please give me some hints or has any experience in such a good > DNase which doesn't leave any DNA traces?! I am not optimistic in that. > > Emad > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From higginsb78 from gmail.com Thu May 22 12:10:50 2008 From: higginsb78 from gmail.com (Brian P Higgins) Date: Thu May 22 12:33:22 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: References: <200805201704.m4KH48O11148@net.bio.net> Message-ID: Have you tried swapping out ALL of your reagents? Many times trace DNA can be found there. Do you use barrier tips for every single procedure in lab, or just your RNA work? Hopefully you have RNA only pipettes, but if not, you can try wiping down the barrels of the pipettes to get rid of any contaminating DNA (it's a bit extreme, but why not cover everything :) Make sure all of your reagents are used for RNA work only as well, as someone else in lab could be contaminating them if they are for general use. Sorry if you've already done all these--they are the basic steps to getting rid of DNA, but your email didn't stipulate what measures other than DNase you've taken. Also, are you detecting the DNA contamination on a gel or in the qPCR? If the latter, what cycle? If it's anything past 28-30 cycles, check the melt cure to make sure it's truly contamination--most negative controls will accumulate enough junk at cycle 30 or so to give you a weak Ct. Good luck, Brian On Thu, May 22, 2008 at 5:17 AM, Emad wrote: > Hello, > > I am doing real time PCR for analysing my genes expression under different > condition of my fungus. We use SYPR green. Actually I isolated RNA using > both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on > column following the kit's instruction. Unfortunately I could still detect > a > DNA contamination!! I treated the RNA again with DNase from NEB but still > some traces of DNA exist. I tried to avoid the DNase treatment because I > learned from some people who deal with RT-PCR to avoid DNase treatment and > used Intron Span primers, and here is the big suffering, I used 18-20mere > primers which locate just in the middle of exon borders and still these > primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and > still > get some amplification, isn't that terrible? I thought to go to 3 bases at > the 3' end of the primer but then you don't get any good prime which suit > in > this area!!? > > Can somebody please give me some hints or has any experience in such a good > DNase which doesn't leave any DNA traces?! I am not optimistic in that. > > Emad > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From crivera from cibnor.mx Thu May 22 10:25:04 2008 From: crivera from cibnor.mx (crivera) Date: Thu May 22 12:33:28 2008 Subject: protein extraction from gel Message-ID: <482EE185@cibmail.cibnor.mx> Hi My name is Crisalejandra Rivera and I?m making my Ph D, right now i?m trying to purifiy an enzyme, i try different methodologies (Chromatography:HIC, GF,IC), now im looking for a protocol to extract proteins from gel, I saw the message on internet about elute protein from gel, could you please send me the refrence or protocol to do it. Today I try to elute my protein from gel using buffer, glycerol... I will incubate overnight and test tomorrow morning. Thanks in advance. atte Crisalejandra Rivera P?rez Grupo de Bioqu?mica Centro de Investigaciones Biol?gicas del Noroeste, S.C Mar Bermejo No. 195, Col. Playa Palo de Santa Rita A.P. 128; La Paz, BCS 23090, M?xico Tel:(+52) (612)-123-84-84 Ext. 3655 y 3656 From sudhee26 from gmail.com Thu May 22 11:47:55 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 22 12:33:36 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: References: <200805201704.m4KH48O11148@net.bio.net> Message-ID: Hey, i have never isolated RNA using columns..but just Trizol and rarely i get DNA contamination. It is surprising that DNAse still not able to digest your DNA i suggest u to run following control lanes in the gel 1. just DNA loading Dye 2. control DNA incubated with DNAse 3. Just DNAse i guess that must trouble shoot it. Sudheendra. On Thu, May 22, 2008 at 2:47 PM, Emad wrote: > Hello, > > I am doing real time PCR for analysing my genes expression under different > condition of my fungus. We use SYPR green. Actually I isolated RNA using > both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on > column following the kit's instruction. Unfortunately I could still detect > a > DNA contamination!! I treated the RNA again with DNase from NEB but still > some traces of DNA exist. I tried to avoid the DNase treatment because I > learned from some people who deal with RT-PCR to avoid DNase treatment and > used Intron Span primers, and here is the big suffering, I used 18-20mere > primers which locate just in the middle of exon borders and still these > primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and > still > get some amplification, isn't that terrible? I thought to go to 3 bases at > the 3' end of the primer but then you don't get any good prime which suit > in > this area!!? > > Can somebody please give me some hints or has any experience in such a good > DNase which doesn't leave any DNA traces?! I am not optimistic in that. > > Emad > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From sudhee26 from gmail.com Thu May 22 12:46:42 2008 From: sudhee26 from gmail.com (Sudheendra Rao N R) Date: Thu May 22 17:01:51 2008 Subject: protein extraction from gel In-Reply-To: <482EE185@cibmail.cibnor.mx> References: <482EE185@cibmail.cibnor.mx> Message-ID: Hi, check this up! http://www.sheer.net/don_article1.htm Sudheendra. On Thu, May 22, 2008 at 8:55 PM, crivera wrote: > Hi > > My name is Crisalejandra Rivera and I?m making my Ph D, right now i?m > trying > to purifiy an enzyme, i try different methodologies (Chromatography:HIC, > GF,IC), now im looking for a protocol to extract proteins from gel, I saw > the > message on internet about elute protein from gel, could you please send me > the > refrence or protocol to do it. > > Today I try to elute my protein from gel using buffer, glycerol... I will > incubate overnight and test tomorrow morning. > > Thanks in advance. > > atte > > Crisalejandra Rivera P?rez > Grupo de Bioqu?mica > Centro de Investigaciones Biol?gicas del Noroeste, S.C > Mar Bermejo No. 195, Col. Playa Palo de Santa Rita > A.P. 128; La Paz, BCS 23090, M?xico > Tel:(+52) (612)-123-84-84 > Ext. 3655 y 3656 > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Think before agree Think before you nod but STOP thinking and You Are God From akhan357 from sbcglobal.net Thu May 22 15:32:28 2008 From: akhan357 from sbcglobal.net (AK) Date: Thu May 22 17:02:10 2008 Subject: DNA EXTRACTION References: Message-ID: after battling with same problem I was finally able to get good quality DNA for any downstream application. methods bellow worked well for removal of sugars and subsequent isolation of DNA, Manning, K. (1991) Isolation of nucleic acids from plants by differential solvent precipitation. Anal. Biochem. 195: 45-50 or/and by using MasterPure Leaf DNA extraction kit (Epicenter Technologies) you can also look at detailed method at http://etd.lsu.edu/docs/available/etd-1027102-193854/ page 49. HTH, AK "kamalaker nasani" wrote in message news:mailman.141.1211256229.3533.methods@net.bio.net... >I am working wth okra(Bhendi). As we all know that bhendi will be having > lot of mucelage. It became a big problem for DNA extration. And llso for > protein extraction for ELISA. Please if anybody have any information > related to this please share with me. > > > Thanx in advance > > -- > URS > Kamalaker Nasani > > > > "The illiterate of the 21st Century will not be those > who cannot read or write. The illiterate will be those > who cannot learn, unlearn and relearn" > -George Bernard Shaw From akhan357 from sbcglobal.net Thu May 22 15:46:28 2008 From: akhan357 from sbcglobal.net (AK) Date: Thu May 22 17:02:15 2008 Subject: IP Protocols and hints References: <4830d6d8$1@clarion.carno.net.au> Message-ID: I recently succeeded in endogenous co-IP of some erythrocyte membrane/peripheral proteins. the protocols and recipes in paper below may provide you with beginning at least. as you know IP is trial and error with different buffers/ detergents/ etc. However, you mention that you want to purify?? this protein by IP? IMHO you may end up wasting a lot of time for unusable amount of protein. AK J Biol Chem. 2008 May 23;283(21):14600-9. Epub 2008 Mar 17. Dematin and Adducin Provide a Novel Link between the Spectrin Cytoskeleton and Human Erythrocyte Membrane by Directly Interacting with Glucose Transporter-1. "Bean Long" wrote in message news:4830d6d8$1@clarion.carno.net.au... > Hi all, > > I've recently got my hands on an antibody to a small protein that's part > of an enormous complex (over 100 nm in diameter) and thought I might try > some IP to purify it. I've also got some Protein G-sepharose beads in the > fridge which I could use for IP. Can anyone offer protocols or point me in > the direction of some to help me out? I'll need to do this under native > conditions. I've done very little IP before, so any hints would be handy. > > Cheers, > > Bean From analia_alet from intech.gov.ar Thu May 22 15:19:31 2008 From: analia_alet from intech.gov.ar (Analia Alet) Date: Thu May 22 17:02:21 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: <200805221704.m4MH44O11468@net.bio.net> References: <200805221704.m4MH44O11468@net.bio.net> Message-ID: <1211487571.4835d553691ba@webmail.intech.gov.ar> Hi Emad, for DNAse treatment I have employed TURBO DNA Free form Ambion (cat # 1907) and it worked well for me. Perhaps your problem is not the DNase. Perhaps you have two many DNA/ ug RNA. This kit, for example, removes up to 50 ug DNA/ml RNA. If there is mor DNA in the sample, the manufactures advise to do a rigorous DNAse treatment, were you use more DNAse, adding it in to steps to the reaction mix and you duplicate the reaction time. Another thing to wonder: are you DNAse working well? Also you can optimize the digestion times with your owun sample and your own kit. Perhaps you need to treat the samples for more time. Remember that kits are standars, and sometimes you have to adjust them to your own conditions. Here it is the protocol we used: Reaction mix: 20ug RNA 10ul 10X Buffer 2ul TURBO DNA-free up to 100 ul water incubate 30 min 37?C add 10 ul DNAse inactivation reagent incubate 2 min at room temperature (mix 2-3 times by inversion) ccentrifugate 15 min 10000g 4?C SN to a new tube Store -80?C Hope it works for you Good luck Best regards Anal?a Mensaje citado por methods-request@oat.bio.indiana.edu: Hello, I am doing real time PCR for analysing my genes expression under different condition of my fungus. We use SYPR green. Actually I isolated RNA using both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on column following the kit's instruction. Unfortunately I could still detect a DNA contamination!! I treated the RNA again with DNase from NEB but still some traces of DNA exist. I tried to avoid the DNase treatment because I learned from some people who deal with RT-PCR to avoid DNase treatment and used Intron Span primers, and here is the big suffering, I used 18-20mere primers which locate just in the middle of exon borders and still these primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and still get some amplification, isn't that terrible? I thought to go to 3 bases at the 3' end of the primer but then you don't get any good prime which suit in this area!!? Can somebody please give me some hints or has any experience in such a good DNase which doesn't leave any DNA traces?! I am not optimistic in that. Emad From ben.long from yourfinger.anu.edu.au Fri May 23 01:44:33 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Fri May 23 11:48:17 2008 Subject: IP Protocols and hints In-Reply-To: References: <4830d6d8$1@clarion.carno.net.au> Message-ID: <483667d3$1@clarion.carno.net.au> AK wrote: > I recently succeeded in endogenous co-IP of some erythrocyte > membrane/peripheral proteins. the protocols and recipes in paper below may > provide you with beginning at least. as you know IP is trial and error with > different buffers/ detergents/ etc. However, you mention that you want to > purify?? this protein by IP? IMHO you may end up wasting a lot of time for > unusable amount of protein. > AK Many thanks AK. I s'pose 'enrich' would be a better word? The hope is to do a bit of proteomic work and subunit relative abundance analysis on it. Enough to run relative pure stuff on a gel would be handy. I had a little play with the Protein G-sepharsoe I found but ended up with bucket loads of what I assume is Protein G on my gels. I also think it's a little too old for successful IP, so I'm getting a new batch! Cheers, Bean From jec73 from cornell.edu Fri May 23 04:55:14 2008 From: jec73 from cornell.edu (Jeremy Coate) Date: Fri May 23 11:48:25 2008 Subject: qRT-PCR and DNA contamination In-Reply-To: <1211487571.4835d553691ba@webmail.intech.gov.ar> References: <200805221704.m4MH44O11468@net.bio.net> <1211487571.4835d553691ba@webmail.intech.gov.ar> Message-ID: <42f327750805230255w15d3bd0co269eea50747b33d9@mail.gmail.com> I would reiterate a point that Brian made earlier in this string - negative controls frequently exhibit some "amplification" in qPCR, but if the cycle number is high it should not be a concern. Even if you do have some residual genomic contamination that is amplifying in the absence of cDNA (I'm guessing you are running controls in which you do not reverse transcribe your RNA), it probably is not contributing to the signal in your cDNA samples. My no-RT controls frequently show some amplification (at high Ct's) that does indeed appear to be from genomic DNA, but when I run the cDNA reactions on a gel there is no evidence of genomic amplifcation. Presumably this is because the cDNA is in much greater abundance than the residual genomic contamination. If the Ct of your negative control is 10 cycles higher than your cDNA samples then the genomic DNA is only about 0.1% of the cDNA abundance. And, as Brian said, you frequently get a Ct value even if there is no DNA template - its not unusual for me to see Ct's of 33 or so for my water controls. But as long as your target templates are amplifying at much lower cycles, whatever this artifactual amplifcation is should not be affecting your results in any significant way. Of course, if you are getting amplification in your controls at cycles similar to your targets, then you do have a significant contamination problem, and the other suggestions posted here apply. Jeremy On Thu, May 22, 2008 at 4:19 PM, Analia Alet wrote: > Hi Emad, for DNAse treatment I have employed TURBO DNA Free form Ambion > (cat # > 1907) and it worked well for me. Perhaps your problem is not the DNase. > Perhaps you have two many DNA/ ug RNA. This kit, for example, removes up to > 50 > ug DNA/ml RNA. If there is mor DNA in the sample, the manufactures advise > to > do a rigorous DNAse treatment, were you use more DNAse, adding it in to > steps > to the reaction mix and you duplicate the reaction time. > > Another thing to wonder: are you DNAse working well? > Also you can optimize the digestion times with your owun sample and your > own > kit. Perhaps you need to treat the samples for more time. Remember that > kits > are standars, and sometimes you have to adjust them to your own conditions. > > Here it is the protocol we used: > > Reaction mix: > 20ug RNA > 10ul 10X Buffer > 2ul TURBO DNA-free > up to 100 ul water > > incubate 30 min 37?C > add 10 ul DNAse inactivation reagent > incubate 2 min at room temperature (mix 2-3 times by inversion) > ccentrifugate 15 min 10000g 4?C > SN to a new tube > Store -80?C > > Hope it works for you > Good luck > Best regards > Anal?a > > > > > Mensaje citado por methods-request@oat.bio.indiana.edu: > Hello, > > I am doing real time PCR for analysing my genes expression under different > condition of my fungus. We use SYPR green. Actually I isolated RNA using > both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on > column following the kit's instruction. Unfortunately I could still detect > a > DNA contamination!! I treated the RNA again with DNase from NEB but still > some traces of DNA exist. I tried to avoid the DNase treatment because I > learned from some people who deal with RT-PCR to avoid DNase treatment and > used Intron Span primers, and here is the big suffering, I used 18-20mere > primers which locate just in the middle of exon borders and still these > primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and > still > get some amplification, isn't that terrible? I thought to go to 3 bases at > the 3' end of the primer but then you don't get any good prime which suit > in > this area!!? > > Can somebody please give me some hints or has any experience in such a good > DNase which doesn't leave any DNA traces?! I am not optimistic in that. > > Emad > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > -- Jeremy Coate Dept. of Plant Biology Cornell University jec73@cornell.edu 607-342-2679 From L.Kamalian from liverpool.ac.uk Fri May 23 05:17:35 2008 From: L.Kamalian from liverpool.ac.uk (Kamalian, Laleh) Date: Fri May 23 11:48:35 2008 Subject: 3 questions Message-ID: <31DC2DFFD70C174B8E3072803F894F91046C89@EVSSTAFF1.livad.liv.ac.uk> I have 3 questions if someone could help me with those please. 1. Could 2 different bands (size wise) be detected for the same protein using poly colonal and monoclonal antibodies? 2. If the samples are run on electrophoresis for too long, can the size of the protein show difference comparing to the previous (shorter) runs? 3. In siRNA , I have knock down my gene of interest at the mRNA level (70-80% according to quantitative PCR x2 experiments), but western blot of the same colony cell protein lysates show no or less ( only 30% ) decrement. Any explanation please? Thanks Laleh From peter.ianakiev from gmail.com Fri May 23 07:34:40 2008 From: peter.ianakiev from gmail.com (peter) Date: Fri May 23 11:48:45 2008 Subject: Hydroshear DNA Message-ID: <5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b@k13g2000hse.googlegroups.com> Hello guys, I need some expertise in random DNA fragmentation. I was thinking of hydroshearing DNA by passing trough syringe. Does anyone know the diameter of the needle needed to shear the DNA? is there better methods? - any experience is greatly appreciated. Thanks, Peter From nick.theodorakis from gmail.com Fri May 23 08:45:51 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 23 11:48:52 2008 Subject: IP Protocols and hints References: <4830d6d8$1@clarion.carno.net.au> <483667d3$1@clarion.carno.net.au> Message-ID: <532739c6-c058-4830-8612-f6db5ab536c2@x35g2000hsb.googlegroups.com> On May 23, 2:44?am, Bean Long wrote: > > Many thanks AK. I s'pose 'enrich' would be a better word? The hope is to > do a bit of proteomic work and subunit relative abundance analysis on > it. Enough to run relative pure stuff on a gel would be handy. I had a > little play with the Protein G-sepharsoe I found but ended up with > bucket loads of what I assume is Protein G on my gels. I also think it's > a little too old for successful IP, so I'm getting a new batch! More likely it was IgG you saw on your gel. The heavy chains are between 50-60 kDa and the light chains are 20-something-ish kDa. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From nick.theodorakis from gmail.com Fri May 23 08:47:06 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 23 11:48:58 2008 Subject: Hydroshear DNA References: <5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b@k13g2000hse.googlegroups.com> Message-ID: <712c8ee2-2d34-4b8a-b774-bcc7e08dc336@c65g2000hsa.googlegroups.com> On May 23, 8:34?am, peter wrote: > Hello guys, > I need some expertise in random DNA fragmentation. I was thinking of > hydroshearing DNA by passing trough syringe. Does anyone know the > diameter of the needle needed to shear the DNA? is there better > methods? - any experience is greatly appreciated. > Thanks, > Peter How big do you need the pieces to be and what size distribution is acceptable? Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From peter.ianakiev from gmail.com Fri May 23 08:56:44 2008 From: peter.ianakiev from gmail.com (peter) Date: Fri May 23 11:49:04 2008 Subject: Hydroshear DNA References: <5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b@k13g2000hse.googlegroups.com> <712c8ee2-2d34-4b8a-b774-bcc7e08dc336@c65g2000hsa.googlegroups.com> Message-ID: On May 23, 9:47?am, Nick Theodorakis wrote: > On May 23, 8:34?am, peter wrote: > > > Hello guys, > > I need some expertise in random DNA fragmentation. I was thinking of > > hydroshearing DNA by passing trough syringe. Does anyone know the > > diameter of the needle needed to shear the DNA? is there better > > methods? - any experience is greatly appreciated. > > Thanks, > > Peter > > How big do you need the pieces to be and what size distribution is > acceptable? > > Nick > > -- > Nick Theodorakis > nick_theodora...@hotmail.com > contact form:http://theodorakis.net/contact.html The distribution is not that important , just I need complete random representation. So far I am doin low complexity libraries, when I go to human DNA, then I will have to get it into the tight range say 0.5 to 2 kb. -Peter From higginsb78 from gmail.com Fri May 23 13:00:30 2008 From: higginsb78 from gmail.com (Brian P Higgins) Date: Fri May 23 13:43:10 2008 Subject: 3 questions In-Reply-To: <31DC2DFFD70C174B8E3072803F894F91046C89@EVSSTAFF1.livad.liv.ac.uk> References: <31DC2DFFD70C174B8E3072803F894F91046C89@EVSSTAFF1.livad.liv.ac.uk> Message-ID: Regarding point 3, you are comparing two methods that differ greatly in their sensitivity. I wouldn't expect them to match up to the same degree. You also need to take into account whatever you are using to standardize/normalize in each case. Assuming a linear relationship and perfect correlation between mRNA levels and protein can be dangerous. On Fri, May 23, 2008 at 6:17 AM, Kamalian, Laleh wrote: > I have 3 questions if someone could help me with those please. > 1. Could 2 different bands (size wise) be detected for the same protein > using poly colonal and monoclonal antibodies? > 2. If the samples are run on electrophoresis for too long, can the size of > the protein show difference comparing to the previous (shorter) runs? > 3. In siRNA , I have knock down my gene of interest at the mRNA level > (70-80% according to quantitative PCR x2 experiments), but western blot of > the same colony cell protein lysates show no or less ( only 30% ) decrement. > Any explanation please? > > Thanks > Laleh > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > From nick.theodorakis from gmail.com Fri May 23 11:57:42 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 23 13:43:17 2008 Subject: Hydroshear DNA References: <5aa1d425-dcae-4ab1-8a6a-4b3d6a0a7e7b@k13g2000hse.googlegroups.com> <712c8ee2-2d34-4b8a-b774-bcc7e08dc336@c65g2000hsa.googlegroups.com> Message-ID: <98a045b4-a61c-4bef-8476-6f4a109f444d@x41g2000hsb.googlegroups.com> On May 23, 9:56?am, peter wrote: > The distribution is not that important , just I need complete random > representation. So far I am doin low complexity libraries, when I go > to human DNA, then I will have to get it into the tight range say 0.5 > to 2 kb. I don't think you'll get it that small with a needle and syringe, but a probe-type sonicator will do it. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From nick.theodorakis from gmail.com Fri May 23 12:01:34 2008 From: nick.theodorakis from gmail.com (Nick Theodorakis) Date: Fri May 23 13:43:26 2008 Subject: 3 questions References: Message-ID: <57353a51-f6e6-4655-aece-c24d9ddaf0f9@y38g2000hsy.googlegroups.com> On May 23, 6:17?am, "Kamalian, Laleh" wrote: > I have 3 questions if someone could help me with those please. > 1. Could 2 different bands (size wise) be detected for the same protein using poly colonal and monoclonal antibodies? Possibly, if there is proteolysis, processing, or alternative splicing. > 3. ?In siRNA , I have knock down my gene of interest at the mRNA level (70-80% according to quantitative PCR x2 experiments), but western blot of the same colony cell protein lysates show no or less ( only 30% ) decrement. ?Any explanation please? The protein might have a long half-life. Or there are two or more isoforms but the siRNA targets only one of them. Nick -- Nick Theodorakis nick_theodorakis@hotmail.com contact form: http://theodorakis.net/contact.html From galahardik from gmail.com Sat May 24 07:52:16 2008 From: galahardik from gmail.com (HG) Date: Sat May 24 14:45:11 2008 Subject: Fishin out possible interacting protein for known RNA Message-ID: <3ca2395b-1e2c-4892-a604-8057a3079b48@z24g2000prf.googlegroups.com> I am currently working on Non Coding RNA I am looking for biochemical approach to find its possible interacting protein so that i can understand more about the function and mechanism of its action. please help Thanks HG From galahardik from gmail.com Sat May 24 07:54:41 2008 From: galahardik from gmail.com (HG) Date: Sat May 24 14:45:26 2008 Subject: In situ hybridization with DNA probe References: Message-ID: <4b156691-f7cc-4148-9a2b-b74cd0560cc6@w5g2000prd.googlegroups.com> hello It is there in Book InSitu Hybridization A Practical Approach series (google it u willl get that!!!) Hope it helps HG From ben.long from yourfinger.anu.edu.au Sun May 25 18:16:48 2008 From: ben.long from yourfinger.anu.edu.au (Bean Long) Date: Sun May 25 20:03:33 2008 Subject: IP Protocols and hints In-Reply-To: <532739c6-c058-4830-8612-f6db5ab536c2@x35g2000hsb.googlegroups.com> References: <4830d6d8$1@clarion.carno.net.au> <483667d3$1@clarion.carno.net.au> <532739c6-c058-4830-8612-f6db5ab536c2@x35g2000hsb.googlegroups.com> Message-ID: <4839f35f$1@clarion.carno.net.au> Nick Theodorakis wrote: > On May 23, 2:44 am, Bean Long wrote: > > >> Many thanks AK. I s'pose 'enrich' would be a better word? The hope is to >> do a bit of proteomic work and subunit relative abundance analysis on >> it. Enough to run relative pure stuff on a gel would be handy. I had a >> little play with the Protein G-sepharsoe I found but ended up with >> bucket loads of what I assume is Protein G on my gels. I also think it's >> a little too old for successful IP, so I'm getting a new batch! > > More likely it was IgG you saw on your gel. The heavy chains are > between 50-60 kDa and the light chains are 20-something-ish kDa. My first thought too Nick, but the same bands showed in my Protein-G only control... no IgG added!! Any recommendations for good sources of Protein-A or Protein-G beads for IP?? Cheers, Bean From engelbert_buxbaum from hotmail.com Mon May 26 07:35:55 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon May 26 11:57:44 2008 Subject: protein extraction from gel References: Message-ID: Am 22.05.2008, 11:25 Uhr, schrieb crivera : > to purifiy an enzyme, i try different methodologies (Chromatography:HIC, > GF,IC), now im looking for a protocol to extract proteins from gel, I > saw the message on internet about elute protein from gel, could you > >please send me the refrence or protocol to do it. This method has only limited applicability for enzyme isolation, as most electrophoretic methods - in particular SDS PAGE - work with denatured proteins. You could however try methods designed to keep the protein happy, e.g. Blue native or CTAB PAGE, the latter of course without SH-reagent in the sample buffer. In principle, you cut the band out of the gel (blue native PAGE is nice as you do not need to stain), homogenize the slice (passage through 27G needle or Dounce homogenizer) and place the bits onto a piece of Muller gauze into a tube with buffer. The end of the tube is closed with dialysis membrane. Electrical current drives the protein toward this membrane. Several manufactureres, e.g. BioRad, offer equipment to do that, check their catalogue. From trondaun from REMOVE.nt.ntnu.no Mon May 26 02:54:11 2008 From: trondaun from REMOVE.nt.ntnu.no (Trond Erik Vee Aune) Date: Mon May 26 11:57:50 2008 Subject: Questions about protein quantification - from the industry perspective Message-ID: Hi, I've recently transferred from academia into industry and have stumbled onto a problem. I need a tag that can be used N-terminally to quantify expression of its fusion partner - ideally in vivo. I was thinking about GFP, or one of its derivates, but I think this protein might be patented. I've tried searching for relevant patents without success. Can anyone confirm that I need a license for the use of GFP (or any of its derivates) for industrial gene expression quantification? What alternatives are there? Can luciferase be used in vivo (will luciferin enter the cells)? Is its use protected? I'd rather not use antibodies since this is expresive, cumbersome and must be done in vitro. Kind regards, Trond Erik Vee Aune From engelbert_buxbaum from hotmail.com Mon May 26 07:47:00 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon May 26 11:57:56 2008 Subject: 3 questions References: Message-ID: Am 23.05.2008, 06:17 Uhr, schrieb Kamalian, Laleh : > 1. Could 2 different bands (size wise) be detected for the same protein > using poly colonal and monoclonal antibodies? Sure. Any post-translational modification that changes size or charge of the protein results in two bands. Unless your (monoclonal) antibody is specific for either of these forms, you see both. > 2. If the samples are run on electrophoresis for too long, can the size > >of the protein show difference comparing to the previous (shorter) runs? If you run the gel too long, you no longer have the front marker dye. Then determination of Rf values becomes impossible and MW determination imprecise at best. > 3. In siRNA , I have knock down my gene of interest at the mRNA level > (70-80% according to quantitative PCR x2 experiments), but western blot > of the same colony cell protein lysates show no or less ( only 30% ) > decrement. Any explanation please? Correlation between [mRNA] and [Protein] is marginal, explaining only about 1/3 of the variability in [Protein]. There is a lot of regulation going on at the level of translation and protein degradation. See for example @article{Gyg-99a, AUTHOR= {S.P. Gygi and Y. Rochon and B.R. Franza and R. Aebersold}, TITLE= {Correlation between protein and {mRNA} abundance in yeast}, JOURNAL= {Mol. Cell. Biol.}, VOLUME= {19}, YEAR= {1999}, PAGES= {1720-1730}, LANGUAGE= {engl} } From engelbert_buxbaum from hotmail.com Mon May 26 07:51:42 2008 From: engelbert_buxbaum from hotmail.com (Dr Engelbert Buxbaum) Date: Mon May 26 11:58:02 2008 Subject: Questions about protein quantification - from the industry perspective References: Message-ID: Am 26.05.2008, 03:54 Uhr, schrieb Trond Erik Vee Aune : > I need a tag that can be used N-terminally to quantify expression of its > fusion partner - ideally in vivo. I was thinking about GFP, or one of > its derivates, but I think this protein might be patented. I've tried > searching for relevant patents without success. Can anyone confirm that > I need a license for the use of GFP (or any of its derivates) for > industrial gene expression quantification? Check with the companies that sells you the vector. Usually the price for the vector already covers a licence to whatever IP rights are attached. If not, they will probably be happy to negotiate your needs with you - against an appropriate fee, of course. From trondaun from REMOVE.nt.ntnu.no Mon May 26 08:12:09 2008 From: trondaun from REMOVE.nt.ntnu.no (Trond Erik Vee Aune) Date: Mon May 26 11:58:08 2008 Subject: Questions about protein quantification - from the industry perspective In-Reply-To: References: Message-ID: Dr Engelbert Buxbaum skrev: > Am 26.05.2008, 03:54 Uhr, schrieb Trond Erik Vee Aune > : > > >> I need a tag that can be used N-terminally to quantify expression of >> its fusion partner - ideally in vivo. I was thinking about GFP, or one >> of its derivates, but I think this protein might be patented. I've >> tried searching for relevant patents without success. Can anyone >> confirm that I need a license for the use of GFP (or any of its >> derivates) for industrial gene expression quantification? > > Check with the companies that sells you the vector. Usually the price > for the vector already covers a licence to whatever IP rights are > attached. If not, they will probably be happy to negotiate your needs > with you - against an appropriate fee, of course. What I need is a tag to be used in our own expression vectors for in vivo quantification of gene expression. I was thinking about GFP but I am afraid that commercial use of this protein is patented