Sma I religation problem

R. P. Grant +44 1 865 221018 rpgrant at molbiol.ox.ac.uk
Thu Jul 13 04:27:48 EST 1995


In article <t.dunn-1207951143090001 at 130.102.68.5>, t.dunn at mailbox.uq.oz.au (Tim Dunn) writes:
> In article <3tv50s$p5g at dingo.cc.uq.oz.au>, mikep at biosci.uq.oz.au wrote:
> 
>> 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
<SNIP>
> 
> Hi Mike, I'm in the next building to you!
> Sma1 is a notorius "nibbler", in that it will eat back ends if you are not
> very careful of the reaction conditions.  Use the bare minimum of enzyme
> in the correct buffer and incubate at 25 degrees C. We have troubles with
<SNIP>-- 
> Tim Dunn

I'd go along with Tim.  We cut for ONE hour only at 25C, then gel purify to 
make sure we don't get uncut in the prep.  We use QIAEX, fantastic.  No 
problems with religations.

Richard

-- 
Richard P. Grant MA DPhil      rpgrant at molbiol.ox.ac.uk
Nuffield Department of Obstetrics and Gynaecology, University of Oxford.
FAX +44 1 865 69141            TEL +44 1 865 221018
http://sable.ox.ac.uk/~lady0266

The fecal material has impacted on the air circulating device.



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