Simple PCR isolation

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Sep 15 15:01:15 EST 2000


Apparently Taq activity is exrememly hard to get rid of (there was a thread
not too long ago that the activity could withstand precipitation, and even
phenol extraction among other seeminly harsh treatments).  So the problem
with just doing a digest directly is that the overhangs generated could be
filled in by the residual Taq activity.  What I've been doing lately is to
run my PCR reaction through a G-50 spin column like I use for cleaning up
sequencing reactions.  While this won't remove the Taq, it does get rid of
the nucleotides, so that fill in won't be a problem.  At least it hasn't
been a problem for me, since fragments I've generated this way have been
perfectly clonable.  Hope this helps.

Mike

>This seems like a rather basic question, but it may save me some time.
>
>After PCR with primers containing restriction sites added on, can you
>immediately digest the PCR fragments prior to purifying by gel
>extraction?  In other words can you avoid an extre gel purification or
>PCR clean up step prior to restriction digest of the fragments?
>
>Thank you.
>

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


---






More information about the Methods mailing list