cjfields at jove.acs.unt.edu
Mon Mar 22 12:58:12 EST 1999
Vellanoweth's Lab wrote:
> I am trying to amplify a gene that is 3000bp long using genomic plant
> DNA and I would like to know what the best times for each step in the
> cycle would be. The annealing temperature of the primers is 57oC and
> the current cycle I use is the following:
> 1. 95oC - 1 min
> 2. 40oC - 4 min
> 3. 72oC - 1 min
> 4. 95oC - 45 sec
> 5. 57oC - 2 min
> 6. 72oC - 1 min
> Then 34 cycles to step 4
> I was told that perhaps a touchdown PCR would be better but I also
> don't know the times for each temperature for that procedure. You can
> reach me here or through email at <cgama at calstatela.edu>. Any help
> would be greatly appreciated.
Well, this depends on the polymerase used. You're extension time is
definitely way too short. If you are using Taq or something close to
it, the time is ~1000bp/minute (or 3 min for step 3). Pfu polymerase
has and extension time of ~500bp/min (or 6 min for step 3). I'm not
familiar with any others out there, but I'm sure if you check the
literature from the company catalogue it should mention these
specifics. Also, many (like New England Biolabs) have accesible info.
on the web).
C. J. Fields
Graduate Student, Dept. of Biological Sciences
The University of North Texas
email : cjfields at jove.acs.unt.edu
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