From owner-celegans@net.bio.net Wed Jun 11 23:00:00 1997 Path: biosci!agate!news.Stanford.EDU!nntp.Stanford.EDU!not-for-mail From: "David M. Eisenmann" Newsgroups: bionet.celegans Subject: Re: Long-template PCR Date: Thu, 12 Jun 1997 09:22:01 -0800 Organization: Stanford University Lines: 25 Message-ID: <33A03039.2969@cmgm.stanford.edu> References: NNTP-Posting-Host: beckman-33-fp-dynamic-189.stanford.edu Mime-Version: 1.0 Content-Type: text/plain; charset=iso-8859-1 Content-Transfer-Encoding: 8bit X-Mailer: Mozilla 2.01 (Macintosh; I; 68K) Wendy S. Katz wrote: > > Has anyone used the Boehringer Mannheim "Expand Long Template" kit with a > thermal cycler other than the fancy Perkin Elmer job for which their > protocols are optimized? I have a Thermolyne Amplitron II and use > thin-walled 0.5 ml tubes. My cycler transits between temperatures fairly > rapidly, so I find the 10 sec. denaturation time in the Boehringer protocol > particularly troubling. I'm interested to hear whether the protocol had > to be modified extensively to work reliably in other machines (I'm going > after a 10kb product). > > Thanks, > Wendy Katz Wendy, I have used the BMB Expand PCR enzyme with MJ Research machines extensively, and have had great results. I always use 94° for 10 seconds as the first step. I have found the annealing temp. to be the one that needs fiddling with (I use 59° 30 seconds as a starting point). I (as well as many others I Stuart's lab) have used it routinely in the 5kb - 20 kb range, and one guy in the lab actually spanned a cosmid gap by long PCR. What's nice about it too is you can clean up the reaction and inject it directly into worms. Good luck. Dave Eisenmann .