detection of small changes in DNA size

Dr. Ted noone at nowhere.org
Thu Sep 7 21:04:04 EST 1995


In article <DEJIGp.32I at uns.bris.ac.uk>, biajm at zeus.bris.ac.uk (AJ. Murray)
wrote:

> I have a 6.4kb vector with EcoRI and BamHI sites 9bp between the cut 
> sites. I would like to be able to detect on a gel if the enzymes are both 
> cutting, either by seeing the 9bp fragment on a gel, or detecting the 
> difference in the vector size. 
> 
> Please send reply to my email address if you prefer
> 
> --
> Alison Murray
> University of Bristol

Cut with one and check for linearization on gel, end label with 32P and
cut with second enzyme, run an aliquot on a 20% acrylamide gel,expose
undried gel and develop autorad. The 9bp fragment should be easily
visible. Cut bands and count for numbers. These enzymes should not be a
problem as both cut like gangbusters with 9bp on the end, if they had
different performance on ends (from NEB catalog) then digest 1h with bad
end cutter then 1h with good end cutter. This makes no difference with
BamH I/EcoR I.
Good luck.

regards,

Ted Michelini
Institute of Molecular Biology
University of Oregon
tedm at darkwing.uoregon.edu



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