XhoI problems/Footprinting

Greg g.dean at uea.ac.uk
Tue Jun 16 07:35:13 EST 1998


Dear All,

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.

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.

Thanks a lot

Greg.
----------------------------
Dr. Greg Dean
Dept. of Biological Sciences
University of East Anglia
Norwich
Norfolk
NR4 7TJ England
Tel.: 01603 593796
Fax.: 01603 592250
----------------------------

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