Degradation time of 3' A overhang in TA cloning

J. David Spafford jspaffor at gpu.srv.ualberta.ca
Sat Mar 19 03:51:56 EST 1994


I have been using Invitrogen's TA cloning kit with success subcloning PCR
products that have only one sized band.  I have added a fraction of the PCR
product into the ligation reaction.  However, instead of one band as before,
I have multiple bands in a PCR product and want to subclone one of the
fainter ones.  Has anyone had any luck purifying gel bands before they
subclone using the TA cloning kit?  I am worried that the 3' A-overhang will
degrade in the PCR product during the purifying process.  Invitrogen tells
me it shouldn't be a problem if I do it quickly.  My preference for
purifying DNA bands is to electroelude and then ethanol precipitate.
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J. David Spafford
Department of Zoology, University of Alberta
jspaffor at gpu.srv.ualberta.ca
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