Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Apr 25 03:43:22 EST 2000
In article <3904C387.DAFAB2FC at mindspring.com>, Karen Roberts
<leehrat at mindspring.com> writes
>How are pcr conditions determined? the pcr rxns I have done in the past
>always was someone giving me the cycling conditions.
Which is probably how a lot of people have done it.
>Is there a formula of some kind? I am having trouble on some pcr and
>I'd like to address this potetial source.
It starts from the design of your oligos.
Try and match the Tm's of whatever you choose, using a primer design
Chooose Tm's of >55C.
I usually put a GC clamp at the 3' end and avoid A's and T's.
Minimise primer dimer and hairpins in the primer, again primer design
program is easiest.
Minimise false priming of either primer elsewhere within the target, in
particular at the 3' end of the primers, again primer design program
takes the work out of this.
Having got the primers designed get them made, straightforward
deprotection and EtOH pptation has worked fine for us for years. We do
however use more purified oligos for kits or controls etc.
Use 1.5mM Mg as standard, 200uM dNTPs, 1.25 u Taq per 50ul, 0.5-1uM
primers, 100ng human xsomal (30-35 cycles), 100ng bacterial xsomal,
23-28 cycles, 1ng lambda, 20-25cycles, 1ng plasmid, 15-20 cycles and
away you go.
For 0.2ml microtubes you can get away on our 9700 and 2400 with 5 secs
denaturation at 94C, 5 secs annealing at whatever temp. our software
advises (Remebering to allow for any altered bases you may have added
for RE sites etc.) and allow 60 secs at 72C for every 1-2kb of target.
5 secs is quite adequate if your block has actually reached the right
temperatures. Remember that RapidCyclers and LightCyclers use 0 secs for
denaturation and annealing. For Pfu allow 2mins per kb and 2mM MgCl2
For GC rich targets you may need Betaine to 1M final and 5-10% DMSO.
Maybe even 95-98C denaturation and a Taq:proof-reading enzyme mix.
For severe AT rich DNA, try an extension temp of only 60C. At 72C you
could be melting the target.
It is very very very rare for us not to get what we want as a clean
single band on a gel, when running 10% of the PCR out.
PS Having given out this secret info., known only to the privileged few,
I will now be banned from the inner sanctum.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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