XhoI problems/Footprinting

Clemens Suter-Crazzolara, PhD un691cs at genius.embnet.dkfz-heidelberg.de
Wed Jun 17 03:17:02 EST 1998


On Tue, 16 Jun 1998 13:35:13 +0100, Greg  said:

>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.

This reminds me of a similar problem that I had with XhoI at least 6 years
back ! I wnated to subclone a XhoI fragment from lambda DNA into the XhoI
site of bluescript. Although all other subcloning worked like a kiss,
this one was simply no go ! I also tested several companies in paralel
and checked on a gel whether I could get ligation into the vector:
finally I ended up using XhoI from Boehringer, which still didn't work
well, but good enough to get that one single transformant that I needed.
Even on this newsgroup, nobody could come up with an idea of what was
going on, and I still haven't got a clue.

My advice to you is to use another approach if possible. If you have to
use XhoI, test as many vendors as possible. Perhaps change the E.coli
strain from which you purify your DNA, as DNA modification may play 
a role.

clemens






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