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Restriction digestion and CIP

CIP Restriction digestion EcoR1 vector

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#1 anna_parker

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Posted 27 August 2014 - 09:09 AM

Hi everyone,

I am preparing an expression vector to eventually express an ion channel in Hek cells.  Right now I am about to ligate the channel DNA into the vector.  I am only using EcoR1 (invitrogen) to digest the vector so i will need to dephosphorylate as well.  My question is, can I run the restriction digestion and dephosphorylation (using NEB CIP) simultaneously in the same tube.  If so, should i use both buffers? or one over the other?

 

Thanks,

Anna



#2 bob1

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Posted 27 August 2014 - 12:46 PM

No - run the dephos step after the digestion. Note that CIP is very very hard to remove from DNA, so, if you can, avoid it and go for one of the shrimp alkaline phosphatases.



#3 phage434

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Posted 27 August 2014 - 01:00 PM

And use as little as possible and for as short a time as possible. SAP or antarctic phosphatase can both be heat killed, a great feature.







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