unwanted plasmid deletions...subhuman

Chris Boyd chrisb at hgu.mrc.ac.uk
Tue Jun 4 10:38:07 EST 1996


Luti Erbeznik (milerbe at pop.uky.edu) wrote:
: Help, dear netters!! Will try to be as concise as possible - this has 
: been several months worth of hassle, and since I've pretty much 
: exhausted all ideas including those of my colleagues, friends and tech 
: services from Stratagene, Life Technologies, NEB and Schleicher & 
: Schuell, this is probably my last attempt to get fresh feedback. 

: I've been trying to construct a genomic library from a bacterium in 
: either pBluescribe, or pKS. Pretty standard procedure, apparently: 
<snip>

The commonest source of problems in procedures such as this is damaged
DNA.  Therefore I would advise you, firstly, to minimize exposure of
DNA to UV while you are purifying it from the gel.  This cannot be
overemphasized!  Even the use of 'safe' wavelength UV light should be
minimized.  The best procedure is quickly to mark the desired zone on
the gel under UV with a scalpel blade (taking only a couple of
seconds), and do the rest of the cutting under visible light at your
leisure.  If you need a photograph, do it of the gel AFTER you've cut
out the band. This will confirm whether or not you have cut out the
correct region. Quantification is best left to the end of the
purification procedure, when you can run an aliquot on a gel.

The insidious thing about UV-type damage is that the DNA still
restricts, ligates and looks great on a gel, but has lost its
biological activity.

The second thing I would do is to omit the phenol/chloroform step.  It
can damage DNA and is best avoided where possible, e.g., certainly
after gel purification.

Best wishes,
--
Chris Boyd                       | from, | MRC Human Genetics Unit
chrisb at hgu.mrc.ac.uk             |  not  |  Western General Hospital
http://www.hgu.mrc.ac.uk/~chrisb |   for |   Edinburgh EH4 2XU, SCOTLAND



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