ligation of PCR products

Robin Beech robin_beech at maclan.mcgill.ca
Tue Oct 8 09:11:53 EST 1996


In article <199610071519.KAA24304 at wustl.edu>, brett at BORCIM.WUSTL.EDU
(brett) wrote:

>>Hi there,
>>
>>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?

Just a foot note to this. I had been trying to use lambda exonuclease to
generate SS DNA from PCR products which requires a 5' phosphate on the
substrate. Surprisingly I found my PCR products disappearing repeatedly
despite the fact that the primers had no phosphate. This did not happen
with all primers but was a regular problem. After a suggestion from
DNAgency (no affiliation) I checked my old sequencing gels from cloned
fragments and an appreciable number had a base or two missing from the 5'
end of the primer which would presumably leave behind a phosphate group,
and cause my problem. Phosphatasing the primers did seem to remove this
phosphate.

Robin Beech
Institute of Parasitology
McGill University



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