Eric.Dufour at cmm.ki.se
Tue May 22 04:03:15 EST 2001
1) Have you checked the distance between the end of your RE sites and the end of yours primers : see on NEB catalog (no affiliation) p
210-211 to have an idea of the digestion efficiencies of enzymes when sites are close to extremity.
Hind III need more than 3 bases or 20h of digest if 3 bases; no way to digest with less than 3 bases between site and extremity of DNA.
NotI needs 8 bases at least AND a 20h digest. With 20h digest and SpeI it should be OK.
2) Why don't you direct clone you Taq PCR product in a TA vector, there is kit for that and it has always been efficient in my hands
when PCR ampli is good (clear band on gel). It's better to gel-extract you band before cloning.
3) I would add a positive ligation control : why don't you try to religate the insert that was already in your vector. It will tell you
if you have a ligase/buffer/the other insert digestion pb.
Hope it helped
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