sequencing of PCR products

John Nash nash at nrcbsa.bio.nrc.ca
Tue Jan 4 17:45:45 EST 1994


In article <2gc4nl$lmn at mserv1.dl.ac.uk>,
Kathy Stupka <kstupka at sunflower.bio.indiana.edu> wrote:
>I was wondering if someone out there could 'lend me a hand' so to speak. I'm trying to sequence a PCR product of approximately 300 bp and I was wondering what
>was the most efficient method to use. It was suggested that I try Cyclist DNA
>Sequencing Kit from Stratagene. Any comments, opinions, or suggestions would
>be greatly appreciated. Thanks.
>
>Kathy Stupka
>Indiana University
>kstupka at bio.indiana.edu.

I just use a (non-cycle sequencing) standard Sequenase ds method.  I
do a PCR.

Then I run the reaction out on a gel, cut out the band and do a
"glass milk" type extraction (this step may not be necessary if you
have a nice single product), and resuspend the DNA in 10 ul TE-8.

Then, I do a standard 35S sequenase (modified T7 pol, or equiv.
whatever) reaction, except I do the ds-DNA alkali denature at 60 deg C
instead of 37... but then, I do all my denatures under those
conditions.

I used to cycle sequence, but I found it wasn't worth the effort just
for one PCR product... sequenase is quicker... I don't let people put
35S in my pcr machine, 32P's a hassle and 33P's too bloody expensive
just to keep around for a quicky sequence.


-- 
John Nash                           (nash at nrcbsa.bio.nrc.ca)
Institute for Biological Sciences,  National Research Council of Canada,
                 Yet another Aussie-in-exile ;-)
      *** Disclaimer:  All opinions are mine, not NRC's! ***



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