small insert ligation

Peter Hohenstein mirihyel at ruly46.medfac.leidenuniv.nl
Fri Sep 6 06:00:59 EST 1996





On 30 Aug 1996, Michael W. Thompson wrote:

> 	    However, it's been my experience that PCR products with restriction sites near the 
> ends (even with 7 to 8 bases stuck on the end to help the restriction digest) are not cut 
> very well with restriction enzymes.  I don't know why. (If anyone out there can find a 
> reason I'd be grateful to hear it ;)

Strange, lately I've been using PCR primers encoding HindIII, BamHI, XhoI
or EcoRI sites with 4 nucleotides extra (normally I use GATC) and each
time it ligated easy. How do you (if at all) clean up your PCR fragments
before digestion? I use standard phenol/chloroform extraction and EtOH
precipitation and it works fine with me.

Peter Hohenstein
Dept. Human Genetics
University of Leiden
The Netherlands





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