Digestion in sub-optimal buffer - (Apr/10/2007 )
I'm trying to do double-digestions where I'm unable to find a perfect buffer for both enzymes. I can find buffers where according to NEB one enzyme will have 100% activity and another will have 75% or 50%. NEB says that because of this additional enzymes or additional time may be needed. My question is how much additional enzymes, or how much additional time? Is there a good rule of thumb?
just incubate a little longer, like 2h instead of one, and not too long as some re tend to develop star activity when not in the appropriate buffer...
or you could always try to digest sequenially, like first the one re in it's buffer, then purify the dna, then the other re in its buffer....
or you could try to start whith the enzyme requiring the lower molar buffer, digest, inactivate re1, add components (like tris, KAc and the like) to make buffer1 into buffer2 and then digest with re2....
I would firts digest with the RE in the "wrong" buffer, let 1 to 2 hours, take a little fraction and check the digestion on a gel, and then I would add the second RE for one hour.
I dont know if there is rule of thumb for this.
I usually use enzyme which cuts efficiently and use its 75% buffer and the enzyme which I am not so sure or have doubt in its 100% buffer. Usually I let the digest go for an extra 30-60 min. One could add an extra 0.5ul of enzyme.
If I have doubts about both enzymes, i would digest them separately.
Thanks for the answers everyone, I'll try these ideas out.
Better late than never. We had a similar probelm and a colleague found this at the fermentas site:
For us it was really helpful.