ligation of PCR products
jlawrenc at vms.cis.pitt.edu
Mon Oct 7 11:46:55 EST 1996
M. Aitchison <m.j.aitchison at ncl.ac.uk> wrote:
> just to make sure: am I right in assuming that PCR products have no 5'
> phosphate groups, and hence need kinase treatment before they can be ligated
> to each other?
typical oligonucleotides used for PCR have no 5' phosphate. i find it easiest
to kinase the oligo prior to PCR.
> can both be done in the same reaction (tube)?
depend on your PCR buffer. i have no difficulty kinasing 20x primer stocks
and using them straight (in kinase buffer) for PCR. the 5% kinase buffer
remaining doesn't affect *my* reaction buffer.
but note also that Taq DNA polymerase adds a dA at the 3' end of the
replicated strand; this 3' overhang will also interfere with ligations. this
base can be removed with T4 DNA polymerase (and its ever-so-handy exo
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