Subcloning dirty PCR products

Emma Macfarlane emma at HERA.MED.UTORONTO.CA
Thu Aug 25 09:58:13 EST 1994


 
Hi German
You didn't mention what restriction enzymes you are using. Have you checked
to see if they will cut close to the ends of linear DNA? I recently spent
a long time trying to subclone a PCR product which I thought I had digested
with Nde I and Xba I only to find Nde I needs a minimum of 7 base pairs after te recognition site to cut efficiently (the appendices in the NEB catalogue are
good for checking this kind of thing) and I had designed my primers to give
only 5.
Hope this is some help. Good luck
Emma



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