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Sequential Restriction Enzyme Digestion

Sub-cloning PCR Purification EtOH precipitation

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#1 Biochem_newbie



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Posted 27 March 2012 - 09:17 AM

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

#2 Curtis


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Posted 27 March 2012 - 07:50 PM

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.

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