From eleonore.buffet07 from imperial.ac.uk Thu Oct 16 06:49:03 2008 From: eleonore.buffet07 from imperial.ac.uk (Buffet, Eleonore) Date: Thu Oct 16 07:36:51 2008 Subject: [Automated-sequencing] T3 and T7 primers Message-ID: Dear Kenneth, Hi I'm a student having a problem with some extra research I am conducting. I have come across numerous definitions as to the function(s) of T7 and T3. Could you please provide your expert opinion as to which are false? To transfer a plasmid to other bacteria Used as a priming site (my personal preference) Can be used to make ssRNA copies of DNA A promoter for cloned DNA expression Viral RNApol promoter Again, many thanks for you time and excellent postings online. Wishing you much Happy sequencing! Love Eleonore xx From pmiguel from purdue.edu Thu Oct 16 09:16:15 2008 From: pmiguel from purdue.edu (Phillip San Miguel) Date: Thu Oct 16 09:20:51 2008 Subject: [Automated-sequencing] Re: T3 and T7 primers In-Reply-To: References: Message-ID: <48F74CAF.5080103@purdue.edu> Buffet, Eleonore wrote: [...] > > Hi I'm a student having a problem with some > extra research I am conducting. I have come across numerous definitions > as to the function(s) of T7 and T3. Could you please provide your expert > opinion as to which are false? > > To transfer a plasmid to other bacteria > > Used as a priming site (my personal preference) > > Can be used to make ssRNA copies of DNA > > A promoter for cloned DNA expression > > Viral RNApol promoter [...] Hi Eleonore, Your post is (at least partly) on topic for bionet.genome.autosequencing so I'll answer. As a sequencing core facility we are frequently bedeviled by these promoters being used as sequence priming sites. I don't find T3 to be much of a problem. But there are many variants of T7. Because some vectors/constructs use one sequence and other use others, we always ask what vector is being used before picking a T7 primer. The problem arises because T7, T3 and SP6 were not originally designed to be sequence priming sites. Rather they are sites where the RNA polymerases of these respective phages will bind prior to initiation of transcription. So, if you look at the T7 genome, you will find that the T7 site is not perfectly conserved. Apparently T7 RNA polymerase is forgiving of these minor difference. However, DNA polymerases nearly always must be primed to function. If the last base of your primer does not match the last base of the priming site, the reaction will generally fail. (Here having a "dirty" DNA prep could actually help. Contaminating nucleases could chew back the priming oligo so it could be extended. Of course the sequence will likely be of poor quality in such a situation. But arguably better than nothing. Anyway, to answer your question... 4 of the 5 possibilities you present are true. "Viral RNApol promoter" is the best, in my opinion, because it describes their primary (evolved) function--the other 3 correct answers are derived characteristics. The answer I can't make fit is "Transfer a plasmid to another bacterium". Though it wouldn't surprise me if, somehow, this one could be true also. Good luck with your "research", Phillip PS Yeah, I know it is bad form to answer what is obviously a student homework assignment. But the traffic in this group is so low I couldn't resist the temptation. From albval from utu.fi Tue Oct 21 06:57:44 2008 From: albval from utu.fi (J. Albert Vallunen) Date: Tue Oct 21 08:35:35 2008 Subject: [Automated-sequencing] TET-labeled markers on a ABI 3130xl Message-ID: <48FDC3B8.3000905@utu.fi> Hi all, We recently obtained a large quantity of microsatellite markers that we would need to screen as soon as possible. The problem is that the dye set of the primers is FAM, HEX and TET and not the usual three-colour triplet FAM, HEX and NED that works fine with the 3130. Since getting nearly a hundred of new NED-labeled primers to replace the TET-ones really isn't an option I was wondering if anyone knows a way to run TET-labeled markers on an ABI3130? Or is it impossible? I gather we would need at least a TET-containing spectral calibration kit / matrix for the procedure to succeed. I know already that the "flourescent amidite matrix standard kit" for the 310 system which is found on the ABI web page has the correct labels, but is incompatible with 3130 due to having multiple peaks. There also doesn't seem to be any other ready-to-use standards to meet the criteria. I've asked also the local ABI tech support for an answer, but they're still yet to provide a working solution. Any help on the matter would be greatly appreciated, Yours, Albert Vallunen -- J. Albert Vallunen, M.Sc., Graduate student Laboratory of Genetics, Dept. of Biology 20014 University of Turku, Finland tel. +358 2 333 7085 Home address: Valaskalliontie 85 23140 Hietam?ki, Finland tel. +358 40 562 8587 From marketing from paa.gtml1.com Tue Oct 21 10:05:44 2008 From: marketing from paa.gtml1.com (Process Analysis & Automation) Date: Wed Oct 22 09:03:04 2008 Subject: [Automated-sequencing] Seminar: Successful Lab Automation at Hinxton Hall Message-ID: PAA Event Invitation You are invited to a seminar event at Hinxton Hall, Cambridge on 13th January 2009 View the agenda http://paa.cgml1.com/processaalz//lz.aspx?p1=059751S2&p=1&cID=0&cValue=1 register your place now http://paa.cgml1.com/processaalz//lz.aspx?p1=059751S2&w=16&cID=0&cValue=1 Date: 13 January 2009 Venue: Hinxton Hall - Cambridge Time: 10am Start Fee: ?40 + VAT Successful lab automation is key to the success of drug discovery, DNA screening programmes, product development and quality control. 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