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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

	http://twod.med.harvard.edu/labgc/estep/longPCR_protocol.html

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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