degenerate primers PCR

[Steven Goldberg]

ds28 at cornell.edu (David B. Stern) wrote:
>
> Does anyone have some tips on using degenerate primers to PCR genomic and
> cDNA libraries.
> I wonder how many different parameters to vary, etc.  My primers were made
> against a degenerate yeast  sequence and will be used on plant DNA.  Some
> of them are apt to form primer-dimers, etc. and misprime  (especially at
> the lower temps. I will need for initial cycyles)  any suggestions on how
> to get what I want?
> 
> Thanks
> Phil Kogan
> phk1 at cornell.edu

First, it may be important to include more primer DNA to take into account
your degeneracy.
I also found that varying the MgCl2 from 1.0 to 5.0 mM in 0.5 mM in-
crements was very useful to improve specificity.
Also be sure to include controls which contain (in separate reactions)
your sense and anti-sense primer mixes--you'd be surprised how many
false positives you can get using only one primer.

Hope this helps.

Steve Goldberg