DIGESTION OF PCR PRODUCTS

MARCEL KUIPER UDHA094 at OAK.CC.KCL.AC.UK
Wed Apr 1 05:46:00 EST 1992


Dear colleagues,


I am trying to introduce restriction sites at the 5' and 3' end of PCR
products, to ligate the digested product in a vector. Until now I've
been unsuccesfull in doing so. At the 5' end I've introduced a KpnI site
with 6 bp in front of it; at the 3' end I've introduced a BglII site with
2 bp extra at the 3' end (should be sufficient according to the New England
Catalog).
After PCR, I treat the products with Proteinase K to remove Taq polymerase
sticking to the ends, and digest the product with the restriction enzymes
and ligate it into a vector digested with KpnI and BglII.
Unfortunately, the transformed ligation product doesn't yield any colonies.
Can someone help me solve this problem?

Thanks very much in advance,


Marcel Kuiper.



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