ligation of PCR products

Jeffrey Lawrence 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 
activity).



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