how to judge the result of BstXI digestion

Jonathan Rees jon at molbiol.net
Wed Nov 21 14:21:52 EST 2001


I agree - there should be a visible difference between digested and
undigested.  I would suggest you should see between 2 and 4 forms of
undigested plasmid (depending on the exposure time, and how far you run the
gel out), the linear digested plasmid which should 99% double-cut will be a
single clear band.  Now, if you cut with a known single-cutting enzyme (I'm
sure pFL series must have a nice MCS, with either EcoR1, BamH1 or HindIII
etc..) you will get a single band which should be visibly larger than the
double cut - but do a long electrophoresis run, at a reasonable rate (ie.
don't blast it at 100V).  The difference may be subtle, but if you have
image analysis software you may be able to zoom in and improve your
judgement.  I always get 2 obvious bands for uncut plasmid pBluescript or
pGreen etc...I reckon I wouldn't worry about the 20bp fragment, it will be
very faint anyway compared to the linear plasmid.  If someone in a nearby
lab has some BstX1 you could compare its cutting efficiency.  Lastly - If
nothing cuts your plasmid, then it could be contaminated with salt from the
purification, in which case, either reprecipitate and wash with 70% ethanol,
or redo the prep.

Jon Rees
Webmaster for - http://molbiol.net - Free Portal for Molecular Biology and
Bioinformatics


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