summary: Degradation time of 3' A overhang

J. David Spafford jspaffor at gpu.srv.ualberta.ca
Mon Mar 28 14:23:15 EST 1994


Thanks to all who responded to my question of degradation time of the 3'A
overhang of the Taq polymerase PCR reaction when using the TA clone kit. 
Having taken the advice to heart, I cut out bands of appropriate size from a
polyacrylamide gel, added a small piece to PCR reactions and added the
reamplified, unpurified PCR reaction (about 1/100 of 1 ul of undiluted PCR
reaction) to the ligation reaction.  Got lots of positive colonies; worked
like a charm!

Others have purified the DNA first using: low-melting point agarose, freeze
and squeeze, miniprep resins, glass-milk, spin-columns etc.  In general,
purification does not appear to affect the final results. Invitrogen techs
have verified to me that the 3'A overhang degrades over time, but is not
disturbed by the purification process.

Thanks once again,
--------------------------------------------
J. David Spafford
Department of Zoology, University of Alberta
jspaffor at gpu.srv.ualberta.ca
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