HELP!! - ExoIII-mung bean digests
kertesz at micro.biol.ethz.ch
Mon Jul 10 13:48:54 EST 1995
I've been carrying out a routine ExoIII / mung bean nuclease digest to
generate nested deletions for sequencing of a Pseudomonas aeruginosa gene
(about 69% GC content). What I see is not the expected time-dependent size
decrease, but the appearance of distinct bands within a smear which includes a
strong band corresponding to the full size construct. This doesn't change much
over ten minutes with ExoIII - the top band becomes less intense, and the
smear becomes smearier. Control experiments with Bluescript gave what I
expected, slight smearing, but a clear decrease in size with time.
Does anyone know if the GC content has an effect on the efficiency of either
the ExoIII or the MBN step? I have gone up with the temperature (to 42C) in
both steps, and have added 10% DMSO to try and destabilize secondary
structures (which perhaps?? might be stopping either of the nucleases dead in
their tracks, leading to the banding I see) but haven't found much difference.
In addition, after ligating and cloning, we find predominantly the full size
Has anyone else had similar problems?
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