David M. Eisenmann
eisenman at cmgm.stanford.edu
Thu Jun 12 12:22:01 EST 1997
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).
> Wendy Katz
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.
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