Partial digest - Addition of Et.Br.

Martin_Messerle Martin.Messerle at rhein-neckar.de
Fri Feb 16 15:09:21 EST 1996


I have a very large plasmid (about 40 kb) and I would like to insert
an oligonucleotide at  a certain site. Unfortunately the plasmid has
another site for this restriction enzyme.
Usually I perform partial digests by titration the enzyme (with good
success). In this case I have no idea how to separate the linearized
form of the plasmid from the undigested form or how to isolate such a
large plasmid from gels.
I remember that some people perform partial digests in the presence of
ethidium bromide. The idea is that after the first cut, the EtBr
intercalates into the linear DNA preventing any further cutting of the
enzyme. Unfortunately I have no protocol nor any experience with this
techniques.
Any suggestions are wellcome.

If you prefer to send me an EMAIL message, please send it to the
following address:
uk691mm at genius.embnet.dkfz-heidelberg.de

Martin Messerle
Department of Virology
University of Heidelberg
Heidelberg, Germany





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