Help!!!Restriction enzyme digestion troubles.
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Fri Nov 10 11:18:00 EST 2000
In article <LAW2-OE458M19VMczi9000001bf at hotmail.com>, the eminent Rodney
Pettway at BIOSCI/MRC Human Genome Mapping Project Resource Centre wrote
> I start out with high molecular weight DNA.
> When I try and
> carryout my digestion it seems as though my DNA is being degraded
> by the enzyme and I have tried two different enzymes.
If the DNA is say human genomic then you won't see discrete bands on a
normal minigel and may not even resolve discrete bands on a larger gel.
For a nuclease test control, try an incubation in the RE buffer but
without the RE. If that is OK then you can rule out nucleases in your
water, DNA and RE buffer.
You can also try adding 1ug of lambda DNA to your genomic digest. If you
get clean discrete digested lambda bands of the correct size with the
genomic 'smear' in the background then your RE is fine and there is
probably nothing to worry about.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
More information about the Methods