digesting DNA with buffer incompatible enzymes - (Jan/08/2007 )
is it best to use a gel purification step between digestions with 2 enzymes that aren't compatible, or is altering the buffer composition preferable?
H
well altering buffers is not good.
I do double digest with both enzymes if one holds only 75% activity
For certain purposes (check minipreps) 50% activity is ok.
Less is not suitable.
I precipitate between sequential digestion. gel purification takes too much time and u may lose DNA in the process.
And as Fred wrote, I only do a sequential digetsion if one of the enz. has less than 75% activity in that buffer.
I would not alter buffer composition.
And as Fred wrote, I only do a sequential digetsion if one of the enz. has less than 75% activity in that buffer.
I would not alter buffer composition.
This is also what I do, an ethanol precipitation in between. I prefer this over gel purification because of the uv-light and loss of DNA.
I add one buffer to another.
So I start with a digest that uses a low salt buffer. After digestion is complete, I add the high salt buffer and proceed with the restriction enzyme that uses the high salt buffer.
For NEB, Buffer, 1,2, and 4 are low salt. Buffer 3 is high salt