In article <[Only registered users see links. ]>, [Only registered users see links. ] wrote:
Their sites are right next to each other, which might indeed be
a problem. It's not competition but the fact that many restriction
enzymes need extra dsDNA on both ends of their recognition sequence.
KpnI is is supposed to cut well (50-100% activity) with just
one base beyond recognition site. HindIII, however, is more
demanding: no cut at all with one base beyond its recognition,
0-20% with two bases, 50-100% for three (according to Fermentas
So, apart from using different enzyme pair or doing restriction
enzyme-independent cloning, your choices are:
1) Do HindIII digestion in NEB buffer 2, then, after verifying that
it is complete, throw in as much KpnI as reaction conditions afford
and hope for the best.
2) Clone something/anything into HindIII site (obviously, orientation
does not matter in this case; any fragment or 20 bp annealed oligos),
then the double cut will be efficient. (And, in the case of large fragment
inserted, double cut will be eay to monitor/verify).