ABI 373 and PCR pdt

[Eric C. Anderson]

In article <Bibbs-2103950856140001 at 192.42.82.96>, Bibbs at scripps.edu (Lisa
Bibbs) wrote:

> Hello all.
> 
> How much PCR product do you use for direct sequencing with the ABI 373
> terminator kit and how do you prepare it

personally, i treat the pcr product using Exonuclease I and shrimp Alkaline
Phosphatase using the method published by USB (which is also where i get
the enzymes).  then i take an A260 reading straight from the reaction and
use what appears to be 750ng of product.  now...i assume that there is a
reasonable amount of interference in the reading of the absorbance due to
the amount of enzyme in the reaction.  so i don't think that there is
anywhere near 750ng of DNA in here, but in my experience it has always
worked as well or better than plasmid sequencing where i use about 800ng of
DNA.

if you'd like more info on the procedure i use, let me know and i'll be
happy to help you.

eric

-- 
eric c. anderson                                
anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110