Semipreparative (10's of micrograms) PCR

Dan Diaz bl275 at cleveland.Freenet.Edu
Fri Jan 24 11:01:58 EST 1992


I am interested in making many micrograms of a stretch of pBR for
use in a nuclease assay.  I have had moderate success using delta
Taq DNA polymerase (USB) by doing 20-30 PCRs (100 uL each) and
pooling prior to workup and purification.

Under the best of conditions using delta Taq v.2.0 enzyme (which
is supposed to be better than Taq polymerase itself) I am not
getting the yield I am calculating I should.  Phaps I have simply
maxxed out the PCR under my conditions, but perhaps there are 
other things which can be tried.  I have already optimized my
enzyme, dNTP, primer, template, Mg, and annealing temp.  I
though of increasing the volume of each PCR to 200 uL but I have
no experience with this.

I would appreciate any feedback from anyone who has made lg amts
of DNA using PCR.
-- 
Dizzy Dan Diaz/Department of Biochemistry and Sushi Consumption
Graduate School of Hard Knocks, Best Western Reverse Yooniversity
ddiaz at cwru.cwru.edu   bl275 at cleveland.freenet.edu    DDIAZ at CWRU
"Neatness is a phenotype of mediocrity"



More information about the Methods mailing list