lambda DNA resists digestion
HARDIES at THORIN.UTHSCSA.EDU
HARDIES at THORIN.UTHSCSA.EDU
Wed Nov 2 15:30:17 EST 1994
Rob Mauk wrote:
> I have isolated lambda gt10 DNA by PEG precipitation followed by organic
> extraction and ethanol precipitation, and find that it is almost
> completely resistant to digestion by EcoRI and all other enzymes that I
> have tested. Proteinase K treatment did not help, nor did additional
> extractions and precipitations. Plasmid DNA digests normally with these
> enzymes, but if the plasmid is mixed with the lambda DNA, it is rendered
> undigestable. Any comments or suggestions regarding the nature of this
> contaminant, or solutions to the problem would be greatly appreciated.
1. Check and see if there's a lot of tRNA in the DNA prep. There shouldn't
be in a lambda prep., but if you leak whole bacteria through the first spin,
you can get it. If this is your problem, you'll see a huge tRNA band on an
agarose gel, and RNAse added to your r. digest will clear up the problem.
2. If it's salt, then you're probably using too much EtOH in the EtOH prec.,
or making it too cold. If you've tampered with the conditions trying to make
a big white pellet, you've optimized the recovery of salt. In this case,
you can clear it out by reprecipitating with correct conditions. Or, dialysis
will work too, especially on a large prep. If you dialyze, go against TE
+ 0.5 M NaCl first, then 2x against just TE. The high salt in the first step
helps lots of kinds of low MW crud get through the bag.
3. Some procedures can leave a load of EDTA in the DNA, although usually
not a phage isolation procedure. If you have a lot of EDTA, your OD 230
will be as high or higher than the OD 260. I clean this problem up by
dialysis as above, although I suppose you could just titrate with Mg.
4. No matter what it is, you may get a quick fix by just increasing the volume
of the r. rxn. and diluting the inhibitor. Try going up 10 x in vol., even if
you have to EtOH prec. the product to get it on the gel afterwards.
5. Lots of commercial kits involving resins or glass beads will clear out
almost anything. If you go this route, make sure you use one specifically
known to work with lambda-sized DNA, since some resins bind DNA that size so
tight that you'll never get it back off.
6. As to the original procedure: If you carry over much sup. from the PEG
precipitation, it can cause this problem. If you're working in a microfuge
tube, it may be worth respinning after taking off the sup. to knock more sup.
off the walls so you can take that off too. Regarding phenol extraction: lore
has it that 1x phenol extraction can let enough crud through to cause this
problem. If you only do 1 x phenol extraction, it's better to sacrifice
a significant part of the aqueous layer than to risk carrying over any
of the interface material. With multiple phenol extractions, on the other hand,
you don't have to worry about this.
Hope this helps.
Steve Hardies, Dept. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu
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