Restriction with pstI - (Dec/02/2005 )
We are trying to clone a 1.5 kb insert(cut with pstI) into a vector of 3.4 kb also cut with pstI only.
We initially standardised the time of incubation for 30 minutes for proper digestion of both the vector as well as insert. But later when we wanted to repeat the same reaction,we got an absolute smear on the gel for both the vector as well as the insert. Later even reducing the time to 20 minutes gave a smear. All components (Water,BSA,Buffer,DNA) are fine.
Can anyone help me out?
could your RE have died? things like keeping it cold can't be overstated enough. Somone might have left it out of the bench for a while, or who knows what could have happened.
try getting out a fresh tube.