restriction digestion of PCR product for cloning

Koen De Smet k.desmet at
Thu Oct 2 09:40:23 EST 1997

Matthew Knight wrote:
> I am having problems digesting a PCR product which has restriction enzyme
> sites placed into the PCR primers, thus they are very close to the end of
> the PCR product.
> Does anyone have any suggestions on how I can be sure that the
> restriction enzyme is going to cut the PCR product. And will the
> restriction enzyme cut with the sequence being so close to the end of the
> product (i.e. about 5 or 6 bases)
> Thanks in advance
> Matthew Knight

The appendix of the New England Biolabs catalogue contains relevant info 
on cutting near the end of PCR  products. 
If you cannot cut, you can first clone the PCR product (without 
digesting) in a vector with blunt ends (or T-A sticky ends for a Taq 
product), and cut the insert out.

Koen De Smet

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___												                 																												___
___     Department of Medical Microbiology																		___
___     Imperial College School of Medicine at St Mary's    ___
___     London W2 1PG																																		     ___
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