Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

Sequential Restriction Enzyme Digestion - (Mar/27/2012 )

Hi there

I am interested in sub-cloning my DNA into another vector.
This is my first time to do sequential digestion so I am a bit confused about the protocol.

The two enzymes that I am using are Bsp EI (NEB Buffer 3) and Psp XI ( NEB Buffer 4).

From my understanding, after the digestion with the first enzyme, I have to do PCR purification? and/or Et OH precipitation?

I dont know whether I am supposed to do both or just one in order to remove the salt and the enzyme. After I understand that, I am not sure as to how much of the product I should be putting into the second restriction enzyme digest reaction.

If anyone could help me out with this one, I would greatly appreciate it!

Thank you


According to NEB double digest online software:

Double Digest Recommendation(s) for PspXI + BspEI:
<*>Digest in NEBuffer EcoRI at 37°C.
This buffer is not supplied with either enzyme.
At least one enzyme has < 100% activity in this buffer, so additional units of enzyme and/or longer incubation time may be necessary.

Alternatively you can single-digest with BspEI first, then run on gel and cut the band from gel, purify and then single-digest with PspXI.