Sma I religation

Andrei Popov ANDREI.POPOV at bbsrc.ac.uk
Wed Jul 12 04:50:43 EST 1995


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Hello everyone,

>I have been having major troubles with religating Sma I cut pGEM.  The vector
>has been cut, and the linear plasmid gel purified using Wizard clean up system,
>but try as I might it wont religate to itself.  The same routine performed on
>the same plasmid with Eco RI works just fine.
>
>Any help anyone has would be greatly appreciated.

I found two ways to overcome this problem:

1. to use as little of SmaI as possible (some manufacturers
   recommend not more than 2 fold excess)
2. include 50 mkm dNTP into restriction mixture.

as approach number two works I assume there is some 3'-5'
exonuclease activity associated with Sma I or maybe some
polymerase is copurified with SmaI.

Andrei




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