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
