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restriction enzyme pcr cloning


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#1 SF_HK

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Posted 17 April 2018 - 08:13 AM

Hi,

Im trying to do pcr cloning of a gene by adding 2 different restriction enzymes to the pcr primers.

The enzymes I plan to use are EcorV and not1 at pcdna3.0 multiple cloning site. Ecorv yields blunt ends while not1 yields sticky ends. Is this possible? Both enzymes are compatible with a single buffer from NEB.

Im new to cloning and would appreciate any advice.

#2 bob1

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Posted 17 April 2018 - 12:54 PM

It should be fine. Note that blunt end cloning is orders of magnitude less efficient than sticky end. Make sure that you have a few bases (3-6 usually) 5' of the restriction site on the primers, so that the restriction enzymes have something to hold onto when cutting.






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