HELP! -- Protecting DNA on a transilluminator?

David F. Spencer dspencer at is.dal.ca
Thu Jul 17 13:35:06 EST 1997


In article <33CB6EE1.916 at abbott.com>, "Earl J. Gubbins"
<earl.j.gubbins at abbott.com> wrote:

> Vern Shellman wrote:
> > 
> > I am having problems ligating my DNA fragments cut out of a gel.  I
> > get variable results that seem to relate  to how long it takes to cut
> > out the band.  The fastest I can see and cut out a band is about 15
> > seconds and by then I suspect there is already significant UV damage.
> > Is there any way to protect my DNA during UV exposure?
> 
> A recent article in Biotechniques (Vol. 21 #5, pp. 898 - 903; 1996) 
> discussed the use of guanosine or cytidine to protect DNA from UV 
> damage.  The authors claim that the addition of 1 mM of either to the 
> gel increases cloning efficiencies by 10-100X by protecting the DNA from 
> damage.  I have started adding 1 mM cytidine (1:100 dilution of a 100 mM 
> stock solution) to my gels, and while I haven't done direct comparisons 
> it does seem to yield significantly more colonies after ligation & 
> transformation.

The other alternative is to use a transilluminator that produces 365 nm UV
(black light) rather than the more usual 302 nm (or maybe even 254 nm)
wavelength.  The longer wavelength is not quite as sensitive in detecting
ethidium bromide stained DNA but is much easier on DNA in gels as well as
on you if you don't wear adequate protection.

You could also run duplicates of your sample, cut the gel in two after the
run and only stain one half.  By piecing the gel back together on the
transilluminator you will know where to cut, but the DNA you recover will
not have been exposed to ethidium and should suffer very little damage on a
302 nm transilluminator (and essentially none on a 365 nm) because
unstained DNA absorbs very little above 300 nm and virtually nothing at 365
nm.

Dave Spencer

-- 
David F. Spencer, PhD
Dept. Of Biochemistry
Dalhousie University
Halifax, Nova Scotia
Canada

dspencer at is.dal.ca
dspencer at rsu.biochem.dal.ca



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