HELP!! - ExoIII-mung bean digests

Burkhard Hassel bhassel at orion.rz.mdc-berlin.de
Mon Jul 10 10:59:27 EST 1995


In article <kertesz.9.000DD114 at micro.biol.ethz.ch>
kertesz at micro.biol.ethz.ch (Michael Kertesz) writes:

> 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.
> Does anyone know if the GC content has an effect on the efficiency
> of either the ExoIII or the MBN step?

GC content: I never had problems with extreme GC rich
areas, but they were not very long (streches of 100-200
bp).
Smear and original size:
I 've had the same problem once and solved it by peparing
new DNA and digest for a longer time with both restriction
enzymes.
The smear comes in most cases from nicked DNA. Exo III can
digest not only from blunt ends or 5 prime overhangs but
also from nicks, so prepare new DNA.
The only explanation for the original size band that I
could imagine is an only partial digest of the 5 prime (or
blunt) enzyme; and I saw this only once.
Hope that helped.

Burkhard Hassel (bhassel at orion.rz.mdc-berlin.de)




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