ssDNA-Exo3 vs Taq??

gubbinse at rand gubbinse at rand
Wed Jun 1 12:49:02 EST 1994


In article <cc37-270594135538 at 132.236.171.164>, cc37 at cornell.edu (CC) writes:
> Hi --
>        I'm going to make ssDNA for use as a non-radioactive probe in
> in-situ hybridizations, and I was wondering what peoples' opinions were on
> using an exonuclease to digest away the other strand as opposed to using a
> single primer and Taq to make ssDNA?  The Taq reaction would give me only
> the region of DNA  I want with little vector sequence, but I'm not sure how
> much ssDNA I would get -- I could certainly get micrograms of ssDNA with
> ExoIII, but I'm concerned about binding of vector sequences to my material
> (I'd rather not have to do the extra gel purification to cut out the
> shorter fragment).
>                                                -- CC

Hello.

Assymetric PCR, with 1 primer, sometimes works and sometimes not; it is very
sequence - dependent.  I suggest you PCR the region desired with 2 primers,
in which one oligo is kinased and one not.  You can then use lambda exo
(available from BRL) to selectively degrade the kinased strand, leaving the
strand with the 5'-OH relatively pure.  This has worked several times for me
for cloning experiments, although I have yet to try it for probes.

Dr. Earl Gubbins, Abbott Laboratories
gubbinse at randb.abbott.com




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