How well does Taq survive in a frost free freezer?
fgrun at med.cornell.edu
Mon Aug 15 20:27:54 EST 1994
First, PCR is not as easy as everyone makes you think.
I'd try some Taq polymerase from another lab with your kit and perhaps
while you're at it a different batch of dNTPs. If it still fails, send
it back to P-E and ask them for a replacement. Rant and rave and throw
your PI's name about liberally. That seems to work for me. :)
1) Primer matching. Tm, hairpin formation, primerdimers, stoichiometry
2) Mg2+ (start with 2 mM; works with most primer pairs).
3) Template concentrations. Do a dilution series; sometimes too much
template is worse than too little.
4) No of cycles. Stay with 30 unless someone tells you different.
5) Program paramters: Start out with 94 C, 1 min; 55 C, 45s; 72 C, 1
min. This should work on most machines, unless you know better.
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