LIGATION OF PCR PRODUCTS

Dr Tan Tin Wee BCHTANTW%NUSVM at ICNUCEVM.CNUCE.CNR.IT
Thu Jul 18 21:48:08 EST 1991


Sometimes if it doesn't go in straight away, we S1 nuclease or
Mung bean it to blunt it (ie remove the nontemplate nucleotides
put in by Taq polymerase beyond the 3'ends).  Just in  case we
didn't get the concentration of these nucleases right and caused
some overshoot into the 3' ends, we flush with Klenow just to make
sure the stuff is blunt.  Then we ligate.

It works for us but not fantastically well.  Maybe we should put in
some hexammine cobalt chloride to facilitate blunt end ligation
when we are cloning a range of blunt end PCR fragments.

Tin Wee Tan
Dept of Biochemistry, National University of Singapore.



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