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 184.108.40.206>, 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
> 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
> 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 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
The fecal material has impacted on the air circulating device.
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