PCR Subcloning with restriction sites

[Susanne Rohrer]

Tony Schountz wrote:

> "David L. Haviland" wrote:
>
> > May basic oligo design is (starting 5'), 6 bases that match the target
> > sequence, 6 for the enzyme site of choice (can vary depending upon
> > enzyme), and then 20-30 bases that match the desired sequence
> > containing no less than 10 G's and/or C's.  PCR away at 64-66'C
> > annealing and away we go...
> >
> > I've had little trouble following these guidelines.
>
> This is rather interesting. What do you think accounts for the requirements of
> several nucleotides 5' to the restriction site?

For more info, consult the back of the NEB catalogue. Very helpful stuff.