UV damage and cloning, was Re: aberrant DNA migration on an agarose gel
cboyd at holyrood.ed.ac.uk
Thu Aug 3 09:59:52 EST 2000
Rob Jordan <rjordan at u.washington.edu> wrote:
:> An alternative (and I think easier) approach is to go ahead and run the
:> with EtBr and then make sure to visualize with a UV source that won't
:> damage the DNA, e.g. a long-wave hand held UV lamp is what I use. I've
:> used this approach for years without any problems cloning. Of course I'm
:> still careful to try to minimize the UV exposure, but I think the
:> EtBr-stained DNA can still take a lot of long wave UV without damage.
: What I've done (after someone suggested it here) is to put 1mmol/L cytidine
: in the running and staining buffer. I don't do that much cloning so I don't
: know how much it actually helps in my case, but it's very simple. Here's the
: Grundemann and Schomig, Protection of DNA during preparative agarose gel
: electrophoresis against damage induced by ultraviolet light. Biotechniques
: 21:989-903 (November 1996).
One can also use guanosine instead of cytidine.
The abstract of the above is:
Preparative gel electrophoresis of double-stranded DNA usually includes
staining the gel with ethidium bromide followed by illumination with
ultraviolet (UV-B) light. In this report, DNA isolated from agarose
gels was found to be a poor substrate for in vitro transcription,
transformation of E. coli and PCR. Inhibition was not caused by
enzyme-inhibiting impurities in the agarose gel, but was induced by a
standard transilluminator fitted with 312-nm tubes. Interestingly, it
was possible to protect the DNA against UV damage by the addition of
cytidine or guanosine to the electrophoresis buffer.
Chris Boyd | from, but \ Medical Genetics Section
Chris.Boyd at ed.ac.uk | not for, / MMC, Edinburgh Uni.,
http://www.ed.ac.uk/~cboyd EH4 2XU, SCOTLAND
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