Problems with double digestion - (Jan/14/2008 )

Hi everybody,

I hope anybody could give me a clue. I am trying to put three different inserts between KpnI and XhoI (two of them) and KpnI and XbaI (the other one) into pYES2, pcDNA3 and one plasmid for Drosophila cells expression. I started doing single digestions of the three plasmids with every enzyme in the conditions I was going to use for double digestion and they look fine (one single band). I did double digestion and after the ligation step I only got the plasmids religated to themselves. I started all over doing the double digestion sequentially and I found out that with 1.5 ug of DNA, 10 U of KpnI does not cut completely after an overnight digestion in any of the three plasmids. I tried the enzyme from another lab and I could not have the plasmids completely cut. The problem to me is that I sequence one of the plasmids to confirm that the KpnI site was there and it was.

Has anyone any suggestion.

Thank you very much in advance.