What is the perfect PCR?

John Dixon jpcd0 at mole.bio.cam.ac.uk
Fri Oct 21 08:13:22 EST 1994


Hi everybody,

I hope this isn't too similar to the FAQ of how do I design primers? but...

I need to design some primers to PCR from a known sequence, across an
unknown cDNA sequence of unknown length, to the polyA. I also need these
to work repeatably on many different cDNAs. 

Since I know some of the 5' sequence I can choose the length/position/GC
richness etc of the upper primer to give me almost any Tm. Because the
lower primer is a polyT I can also lengthen/shorten the polyT primer to be
compatible with any upper primer.

So, my question to all you PCR guru's is: What is the ideal annealing
temperature for a optimal PCR reaction, seeing that I can choose any Tm
for both my primers from around 44C to 88C? 

Am I right in thinking that Taq works best at 72C, so I should not anneal
any higher and that the lower the annealing temperature is, the more
nonspecific primer:template binding occurs - ie the optimum annealing temp
is 72C?

Or as someone in my lab suggests, to minimise nonspecific binding I should
have primers with low Tms, say 45C, and run a series of PCRs with varying
annealing temps from 72C - 40C, to find the highest temperature at which I
get one (the correct) band, on the grounds my primers will be bound
nowhere but the intended site?

Does any of that make sense? I look forward to finding out. Thanks,

-- 
John Dixon                     Lab 44 (223) 334131
Wellcome/CRC Institute         Fax 44 (223) 334134
Dept Genetics
Cambridge University    
United Kingdom       e-m: jpcd0 at mole.bio.cam.ac.uk



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