HELP!! - ExoIII-mung bean digests

[Michael Kertesz]

Hi everyone,

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 
construct again.
Has anyone else had similar problems?



Michael Kertesz

ETH-Microbiology,
ETH-Zentrum/LFV,
CH-8092 Zurich, Switzerland

tel: +41-1-632 3357
fax: +41-1-632 1148
e-mail: kertesz at micro.biol.ethz.ch