What is the perfect PCR?

[Stuart Brown]

In article <jpcd0-2110941415400001 at macr1-3.welc.cam.ac.uk>
jpcd0 at mole.bio.cam.ac.uk (John Dixon) writes:
 
>
>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
 
 
We always try to design our primers to anneal at 60 to 65C.  But even
in a batch of primers designed with the same parameters, we have to
optimize the conditions for each one individually - ie. the Primer
Design software has a long way to go before we can accurately predict the
behavior of each primer pair in real PCR conditions.
 
 
 
Stuart Brown                       |   Plant Genetic Resources
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