For double sequential digest: which enzyme first? - (Jan/18/2008 )
I'm cloning a PCR product into a vector by using 2 restriction enzymes. I'm doing a sequential digests with an agarose gel/spin column clean up between digest 1 and digest 2 (also the same clean up before ligation)-----the enzymes I'm using are Pst1 and Xma1 (NEB) however, a postdoc recommened I do the Xma1 (buffer 4) digest first then the Pst1(buffer 3) but i don't see why there should be a difference-----can anyone come up with a reason?
Well, if you did the XmaI digest first in the low salt buffer 4, you can then simply add additional high salt buffer 3 and PstI to the digest, without needing to remove the original low salt buffer 4 or clean the digest in between.
It is a short cut, for sequential digest when one enzyme works in a low salt buffer and the other in a high salt buffer.
There is also no need for a clean up between the XmaI and PstI digest. Yes, you have to clean up your PCR product prior to restriction digest and after the sequential digest. But in this intance there is no need to clean up between digest.
PstI cuts so well, in practically any buffer, that I'd just put it all in together.
I've never done a digest where you just add a different buffer and enzyme-----would this be ok? start with a 30ul reaction (3.0ul buffer 4, 0.3ul BSA, DNA, 1.0ul Xma1 enzyme, ddH2O to 30ul-----digest 2-2.5 hours) then take that 30ul and use it in a 50ul reaction (2.0ul buffer3, 1.0ul Pst1 enzyme, ddH2O to 50ul-----digest another 2-2.5hours)
to digest 1ug DNA, 1 Unit enzyme is enough to digest it in 1 hour so you can reduce your digestion time by preparing your sample for digestion with proper calculation
I repeat. Do a double digestion, it will just work. You are making work, using supplies, and reducing your yield if you insist on doing a sequential digestion with these two enzymes.