Megaprimer PCR

Richard V. Giles giles at liv.ac.uk
Thu Dec 10 06:53:16 EST 1998


Csaba Jeney wrote:
> 
> We are trying to get a PCR product using a very long primer pair (60bp
> each, 20 bp anealing length) but until now there is nothing to glad.
> We already have changed the annealing temperature, the Mg
> concentration and the next step will be the primer concentration.
> Could anybody out there suggest other things and/or suggest the ranges
> of these variables to get of rid this problem.
> 
> Thank you in advance.
> 
> Csaba

My experience with such primers (I use them to introduce phage RNA pol
sites) is that they behave just like shorter primers (missing the pol
sequences).

Just in case, denature your primers (95C 10 mins then place on ice)
before use.

Play with the extension time, the denaturation temperature and the ramp
down from denaturation to annealing temperature. I've found that 95C
1min (temperature of thermocouple inside tube) works well with most
polymerases and that the fastest ramp works best.
-- 
Richard V. Giles Ph.D.
School of Biological Sciences
University of Liverpool
Tel +44/151 794 4389
Fax +44/151 794 4349
http://www.liv.ac.uk/~giles/



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