HUM-MOLGEN archive: BIOT: PCR sequencing, RT PCR etc

This message contains three posts relating to various BIOT topics, including PCR sequencing and RT PCR: Martin Kennedy Arthur bergen BIOT Topic Editors, HUM-MOLGEN 8< ======================= cut here ======================= >8 RE>BIOT: various = 11/3/95 I can't immediately offer any help but BioTechniques magazine might. = They offer a Online Library of their techniques journal. I know that in = the past their have been articles which might be just what you're looking = for. Their Telnet address is BIOTECHNET.COM with a username BIOTECH and = a password TECHNIQUE. If someone else from the list doesn't come up with = anything, try them OR I also belong to a list that does forensic work and = I bet that they would be helpful. I will forward your request on to them = if you want. Good Luck! Melissa Caimano University of AL at Birmingham Dept. of Microbiology 8< ======================= cut here ======================= >8 Subj: Re: PCR Sequencing Reply to: RE>PCR Sequencing Arthur, Re: PCR Sequencing We routinely use the Sequenase PCR Product Sequencing Kit from USB. This = kit takes dsPCR products and treats them with Exonuclease III and Shrimp = Alkaline Phosphatase. The treatment only takes about 1 hour total time = then you are ready for normal dideoxy sequencing. The kit costs about = $300 or so but comes with nucleotides, ddNTPs, all buffers and enzymes = excluding Taq. We have had good results with the kit but it has what = appears to be a limitation, at least in our hands, in that the reads tend = to be shorter so I wouldn't recommend this method if you want to try to = read over 275-300 bp per primer/template. If you would like to know more = about this kit or other methods that do not involve kits that I have = used, don't hesitate to call or e-mail. I would be more than happy to = help in any way. Melissa Caimano Univ. of Alabama at Birmingham Dept. of Microbiology (205) 934-9530 melissa_caimano@micro.microbio.uab.edu 8< ======================= cut here ======================= >8 For Peter, The Gentra Purescript kit works well for RNA isolation for RT-PCR at least for BCR-ABL and t(15;17), short transcripts. We are not sure it is sufficient for large transcripts (>2 kb). The cDNA synthesis is most efficient when primed with random oligos; then add the specific amplimers before the PCR step. We are still comparing different RNA kits and/or methods of specimen delivery (whole blood and marow) to optimize the recovery and RT-PCR for larger transcripts. Any suggestions would be appreciated. Barb Zehnbauer