Digestion Problem with BsmBI - (May/05/2006 )
This is my first post (probably of many), so; Hi
I am having trouble digesting my plasmid with BsmBI, I have never used this enzyme before and neither has anyone else I know.
OK, here's the problem, when I digest with BsmBI alone it works but when I do a double digest with XhoI something goes wrong and the gel shows a strange patchy sort of band. The XhoI cuts fine too, when alone.
I was wondering if anyone else has had trouble with this enzyme, and can give me any tips.
What temp. are you digesting BsmBI? I think its 55C.
Also you cannot digest with BsmBI for a long time. try to limit the amount of time you are digesting with this enzyme.
ok few questions to start :
did you try a sequential digestion?
do you digest with both enzymes? so in that case do you do a *2step digestion or 1time digest? I mean by a 2 step few h at 37 and then get to 55° (you can use a pcr apparatus for doing that overnight)?
what buffer do you use?
Thanks for your replies, we gave up on BsmBI and are now using another enzyme which is working fine