Strange problem with digestion and dephosporylation of PCR product - (Jan/19/2011 )
To get 2 PCR products I used two sets of primers with BamHI and Not1 sites and I added extra 6 nucleotides to each primer. After PCR I got my two products exactly the size I wanted (+-1000bp) and no other products. When I digested them with BamH1 and Not1 (1hr, 37 celsius degrees, buffer suitable for both), dephosphorylated them (CIP phosphatase) and purified on gel I got two bands for both products- one the same size as PCR products and one about 50-100bg bigger... Does anyone have any idea? I purified both products and I tried to ligate them twice but my ligations didn't work (I didn't get any colonies).
Dephosphorylation is usually performed on vectors rather than inserts. If your vector also was phosphatase-treated, the fragments cannot ligate. Did you perform any controls?
Also note that NotI requires a minimum of 9bps of overhang for efficient digestion. With only 6bp the efficiency is only 10% even after an overnight digest. So this too will be a contributing factor