Low yield with Taq from GIBCO BRL.

Peter Nilsson Peter.Nilsson at klinkemi.umu.se
Thu Apr 17 03:13:48 EST 1997


Hello.

There are numerous ways of increasing the yield in a PCR reaction. I usually try to optimize the reaction (the primer/template combination in 
question) for the parameters usually described. A good way of doing this is to use a serial dilution of templat diluted ten fold and determin 
the highest possible dilution where you still amplify the template to visible amounts on gel. One can then in a controled way determin the 
effect of each of the parameters one want to optimize. The annealing temp is, for exampel, important. Decreasing it migth have a positive 
effect on yield but migth also have the oposite effect due to increased non-specific amplification. Concentration of primers, MgCl2, 
nucleotides, Taq enzyme migth also influence the yield. Also the cycle numbers migth have a good effect, but again, to many cycles migth 
increase non-spec. ampl. Of course the template conc is important but as I understod you couldn't change that. We are cureently amplifying very 
old samples of paraffin embedded tissue. To increase yield we simply reamplifyed the product once more with the same primers and got product 
from some "impossible" samples. The problem with that is that also non-specific products are amplifyed in the second round since they have a 
perfect primer match. To avoid that one can do a nested PCR. 

About enzymes I don't think there is nay big difference between the different companies Taq enzymes. But there are some other enzymes on the 
market. We have, for example, very good experiences when it comes to yield with Boheringer Mannheims "expand" enzyme mix. Migth be worth 
trying.

Many words, I hope a helped you somewhat. Good luck- Peter Nilsson.
-- 
Peter Nilsson, Molecular Biologist
Clinical Chemistry Department,
Umeå University Hospital,
S-901 85 Umeå,
Sweden.



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