Dennis J. Templeton
djt2 at po.CWRU.Edu
Mon Nov 18 17:35:13 EST 1991
In a previous article, penas at ucbeh.san.uc.edu () says:
>I have yet another question about PCR. What is the minimum GC% that a PCR
>primer should have. What is the maximum and minimum effective size for a PCR
>primer. Thanyou very much, in advance, for your response.
>Sandra Pena de Ortiz : penas at ucbeh.bitnet
>University of Cincinnati
>College of Medicine
Sandra... the answer to your question is 0%
but joking aside, we have found it handy to STANDARDIZE our primers,
so that all work at about the same annealing temp, etc. We have chosen OUR
standard at 22 NT with 11=G+C, then pick our primers to match these
parameters. In rare instances, if we cannot get enough g+c's, we make the
primers one nucleotide longer for every (G+C)<11
If you calculate the melting temp as 2*(A+T)+4*(G+C) then all of these
primers will have a Tm of 66 degrees; in actuality we anneal for 1 sec at
45 degrees routinely, then extend at 72 for 30 secs to 2 mins, depending on
length of product.
Having all of our primers with the same Tm means we can usually do all the
experiments in the same thermocycler run.
More information about the Methods