Where to find tips and advice for design of primers?

Keith Robison robison at mito.harvard.edu
Wed Nov 29 12:05:00 EST 1995

Bo Servenius (bo.servenius at wblab.lu.se) wrote:
: Dear Netters!

: I am trying to collect advices for manual design of primers. Is there any
: good netsites out there with this info or could you help me post some
: here.

: I have an old one regarding purin content - it should not be more than
: 50% - but is this really true nowadays with pure synthesis chemicals.

: Another one is you should have Gs and Cs in the 3'end but not more than 3
: Gs in a row.

: Than thera are the obvious ones regarding secondary structure and
: serlcomplimentation.

In collaboration with 2 colleagues, we've successfully used long PCR
conditions to amplify templates up to 10Kb and the primers work pretty
routinely on E.coli genomic DNA.  You can find the PCR protocol at


which includes our primer picking rules:

	20-23 bp
	12 G+C
	8-11 A+T
	Tm of 60 to 68 degrees
	Hairpin Tm of <= 35 degrees
	No more than 3 G's in a row
	<4 bp of 3' complementarity between primers

To be honest, we do not know whether these rules are excessive;
we just know that they have worked for us routinely using the
protocol at the above URL.  I know, for example, that some 
primer pairs slipped through which violated the last rule, yet
still worked.  Also, I've been told by some that the 
"no more than 3 G's in a row" is probably no longer a problem for 
synthesizers, but it never hurts to be safe.

You might also look into how the big PCR-based STS groups
(Whitehead, CHLC) pick their primers, though I think they basically
just use the MIT Primer program.

Hope this helps

Keith Robison
Harvard University
Department of Cellular and Developmental Biology
Department of Genetics / HHMI

robison at mito.harvard.edu 

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