ligating

KMW4 at psuvm.psu.edu KMW4 at psuvm.psu.edu
Thu Jul 29 17:17:48 EST 1993


I want to attach double-stranded oligos to a mix of DNA fragments.  Then, I wan
t to amplify these fragments by PCR using primers to the attached adapters.  To
 prevent insert chaining, I would like to phosphatase the fragments first, then
 ligate with the double stranded adapters.  The adapters can be synthesized wit
h phosphorylated 5'nucleotides.  But the ligation, using T4, will only covalent
ly attach the phosphorylated nucleotides in the adapter to one of the strands o
f the fragment; the other strand, having been phosphatased, won't ligate.  I un
derstand that when this is done in plasmids, the E. coli cloning bacterial enzy
mes repair the unhooked strand.  But how can I do this for PCR?  Will Taq read
or amplify through a break?  Would Klenow treatment do what is needed?  Please
reply by email if you have tried this or know what will work.  Thanks.

Ken Weiss, PhD  Dept of Anthropology  Penn State University



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