XhoI problems/Footprinting

suraj Mukatira suraj at omni.cc.purdue.edu
Wed Jun 17 11:06:53 EST 1998


un691cs at genius.embnet.dkfz-heidelberg.de wrote:
> 
> On Tue, 16 Jun 1998 13:35:13 +0100, Greg  said:
> 
> >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.
> 
> Re-reading your message, I would presume that your DNA is contaminated
> with something in your agarose. This something is co-purified with
> with the DNA with your qiaexII. Can you change to a higher grade
> agarose ? can you skip the gelpurification step ?
> 
> clemens

Another possibility:

	During the final stage of QUAEXII extraction make sure the pellet is
dry.
May be it is the carry-over ethanol which is causing your digestion
problems. ?


suraj



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