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SAP dephosphorylation


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#1 LacquerHead

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Posted 18 April 2013 - 04:30 AM

Hi all,

Wondering at what stage SAP is best used, following gel purification of the vector cut by a single enzyme, I have done it right after digestion with BamHI and have been having trouble getting a ligated product with my insert, guessing that Im getting recircularization of the vector?
Thanks.

#2 bob1

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Posted 18 April 2013 - 02:09 PM

Unfortunately phosphatase treatment often makes more problems that it solves as it is very very hard to denature and remove. However, in your case it is pretty much essential, so you can't avoid it.

The best time to do it is immediately after digestion. If you use about 1/10th of the recommended amount I find it is easier to clean up.




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