XhoI problems/Footprinting

Darren A. Natale dnatale at box-d.nih.gov.nospam
Tue Jun 16 09:01:23 EST 1998


Greg wrote:

> I am having the most ridiculous problems in preparing large amounts of
> small DNA fragments for DNA footprinting. I am trying to bulk up a 340
> bp fragment from one of our well used plasmids. The method is simple,
> cut SmaI, isolate by agarose gel purification a 2000bp frag (by qiagen
> qiaexII), cut XhoI, and agarose gel purify again, pulling out a 340 bp
> band. Sounds straight forward. I cannot get complete digestion with
> XhoI (~50%), which is dramatically reducing my yield. I have used
> different plasmid preps, different batches of XhoI (all well within
> their shelf life), with very extended incubation times, all  to no
> avail. All the digest conditions are perfect for the enzyme, and
> sufficient enzyme is being used. To top it all my qiagen qiaex is only
> working to about 60% recovery.

I presume that you would have a co-migrating ~340 bp fragment if
you don't do the two-step purification?  To at least eliminate one of
your
60% losses from the qiaex, could you instead cut with Xho I and another 
RE that digests the contaminant band?  Then you would only have to do
one 
round of purification.  Also, could you instead use beta-agarase (NEB)
to recover
your DNA from a low melt gel?  In my hands, I routinely obtain >90%
recovery
with this method (it mostly depends on how much of the band you cut).  I
have
not tried qiaex so I don't know whether the recovery you get is typical
or 
your own bad luck  :).  Another alternative is to purify from
polyacrylamide.
Using a crush and soak method, close to 100% recovery is possible.

> If you have any ideas about my XhoI problems or suggestions for
> purifying large amounts of small fragments of DNA from plasmids for
> footprinting, I would be interested in hearing them.

No explanations, other than to say that I too have had difficulty with
Xho I
(different lots, different companies, etc.).  But here are two
suggestions.
First, try a different enzyme.  Pae R71 (NEB) cuts the same site as
XhoI. Or
use a different specificity altogether.  Maybe the result would be
better 
with a different enzyme.  Second, could you use PCR to amplify the
fragment?
If fidelity is an issue, use Pfu or Pwo.  I cannot tell whether you need 
purified DNA and will footprint after binding in vitro or need to use
methods
that will not disrupt a pre-formed complex.

D. Natale
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