need help with cloning by PCR

David N. Levy david_levy at dfci.harvard.edu
Tue Aug 29 14:21:48 EST 1995


In article <40trdv$80f at mule.fhcrc.org>
lbonin at fred (Lise Bonin) writes:

> To all pcr wizards:
> I'm trying to pcr a 3 kb fragment from a plasmid - with no luck to date.  
> My primers are 30-mers with 14 nt complementarity to the target 
> sequence.......

It sounds like you have tried enough conditions to now question whether
your primers were made correctly.  You have probably double checked the
sequences you wrote on the order sheets for typos, so there may be a
synthesis error.  Try having the primers remade.  Check your primers on
a sequencing gel.

Also, I do not use identical "Clamps" on my primers for fear that they
may anneal to each other too strongly to work.  Since the clamps are
ate the 5' ends you won't get extension (primer dimers) but they might
out compete the template for binding.  I put ggcc on one primer and
gcgc on the other.


David N. Levy
Dana-Farber Cancer Insitute
Boston, MA 02115
david_levy at dfci.harvard.edu



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