no ligation of compatible fragments?

John K. Troyer jtroyer at umabnet.ab.umd.edu
Sat Oct 14 09:45:45 EST 1995


On Fri, 13 Oct 1995, Michael Szardenings wrote:

> Hi,
> I have had some problems cloning fragments into a vector. Finally I
> checked some restriction enzymes by trying to cleave and religate lambda
> DNA. This did not work for our SmaI and BamHI, but worked well for other
> enzymes like HindIII etc.
> SmaI might be artefact, although I have done blunt end ligation under
> similiar conditions before, BamHI is definitely not and SmaI/BamHI is just
> the cloning I am trying. Enzymes are from BRL, so not just 'some'
> supplier, but they have spent a while in the freezer. Nevertheless, I have
> never seen something like this.
> Can enzymes really get 'unspecific', i.e. give different types of cuts on
> the same recognition sequence?
> 
> All ideas are wellome
>                         Michael




Michael:

How long are you digesting and how much Bam are you using?   Bam HI has 
star activity which may cause problems if you either digest for too long 
or your RE concentration is too high.


Regards,

John

***************************************************
John K. Troyer, PhD
University of Maryland School of Medicine
Department of Biological Chemistry
jtroyer at umabnet.ab.umd.edu
***************************************************




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