The big trick with PCR is to get a protocol that works for you. Perhaps the
most important things to consider are magnesium concentration and annealing
temperature.
Most of the protocols I use involve 15 second denaturing and annealing
steps, and 1 minute extensions. The 1 minute extension time is long enough
to amplify fully 1.5kb of 16S rDNA, so I am not to sure about the
1000bp/minute rate for Taq. I do agree that for 3000bp, your extension time
is a little short however.
Advice would be to drop the annealing temperature to something quite low
say 48degrees, and optimise Mg concentration. Then after finding optimal
Mg, increase the annealing temp until you are only amplifying the bits you
want.
Good luck! PCR is more black magic than science!
Paul
>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).
>>Good Luck!
>--
>C. J. Fields
>Graduate Student, Dept. of Biological Sciences
>The University of North Texas
>Denton, TX
>>email : cjfields at jove.acs.unt.edu>\\\\\\\\\\\\\\\\\\\\oooooooooooooooo////////////////////
>"Giving money and power to government is like giving
> whiskey and car keys to teenage boys"
> -P. J. O'Rourke
>"Join the military. Travel to exotic places, meet
>exciting people, then kill them"
> -Anonymous
>////////////////////oooooooooooooooo\\\\\\\\\\\\\\\\\\\\
-----------------------------------------
Paul Taylor
Department of Microbiology and Immunology
University of Melbourne
Parkville Victoria 3052
AUSTRALIA
p +61 3 9344 5698
f +61 3 9347 1540
e taylorpm at ariel.unimelb.edu.au
