pDBleu vector - restriction problems (Jul/13/2006 )
i have been having problems with pDBleu vector intowhich i am planning to subclone a DNA piece. the problem is, i just cannot digest the vector with a second RE. enzymes are both working, i am sure of that. i digest the vector with either sal i or not i , gel purify them (using ultra pure agarose and eluting in water) and cut it reciprocally with these enzymes then again. but i always get self ligation of the vector only (1:3, 1:5 and 1:8 v:i ratios all gave the same result ). pDBleu is 9903bp (a bit long), does this matter?
is there anyone else having similar problems with their vectors or with pDBleu vector?
thanks in advance
I dad some problems with digestion of salI, but then I tried to digest it for longer with SalI. After 3 tries, I managed to get the ligation.
Ofcourse I had some self ligated plasmid but my ligation plate had atleast 2x or 3x the amount of colonies than control. So it was fine eventually.