undigestable genomic DNA

[Ed Siefker]

So I've been extracting mouse tail DNA for use in southerns for some
time, and I keep running into DNA that won't digest. I use a pretty
typical method. I digest the tail piece with Proteinase K at 55C
overnight in a buffer with 100mM NaCL and 1%SDS with some tris and EDTA
as well. Then I extract with an equal volume of
phenol/chloroform/isoamyl alcohol, followed by an extraction with just
chloroform. Then I add 1/10 volume of 3M NaAc, 2 volumes of 100% EtOH
and centrifuge. Wash with 70% EtOH, dry, and resuspend in 10mM tris.

Frequently this DNA is very hard to resuspend, has a very high A230
reading, and does not digest. I gather this is polysaccharide
contamination because the DNA is very globby and rather like snot. I
have read that reprecipitation from 1M NaCL or 2.5M NH4Ac reduces
polysaccharide contamination and it does make the DNA less globby and
reduces the A230 reading, but I still cannot digest it.

Any suggestions would be most appreciated.

[Tom Knight]

Ed Siefker <ebs1...@creighton.edu> writes:

You might try a second round of phenol/chloroform, or (if it really is
polysaccharides) isolation using CTAB instead of SDS. See Current
Protocols.

[Ranadheer Kumar Gupta]

Hi all,
I too got similar kind of problem, but it is with the plant leaf material.
Can it be reduced by Urea purification?
If any of you have some tips, please help me.

regards
Ranadheer

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[Jayakumar, R]

Plant DNA is tough and depends on the type of plants. Monocots are easy
and much cleaner, but dicots are just so difficult, especially if the
plant is an aromatic or an oil seed variety.
Could try usin N-Lauryl sarcosine to clear up the polysaccharides and
PVP for clearing up the polyphenolics. Look for a protocol that uses
these reagents.
Jayakumar

regards
Ranadheer

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[AK]

Dear Ranadheer, I had a tough time with strawberry leaf DNA during my Ph.D,
but finally was able to get good quality DNA for any downstream application.
methods bellow worked well for removal of sugars and subsequent isolation of
DNA,

Manning, K. (1991) Isolation of nucleic acids from plants by differential
solvent precipitation. Anal. Biochem. 195: 45-50

or/and by using MasterPure Leaf DNA extraction kit (Epicenter Technologies)

you can also look at detailed method at
http://etd.lsu.edu/docs/available/etd-1027102-193854/ page 49.

HTH,

AK

"Ranadheer Kumar Gupta" <ranadhe...@gmail.com> wrote in message
news:mailman.263.119271...@net.bio.net...

[Kyle Legate]

Ed Siefker wrote:
> So I've been extracting mouse tail DNA for use in southerns for some
> time, and I keep running into DNA that won't digest. I use a pretty
> typical method. I digest the tail piece with Proteinase K at 55C
> overnight in a buffer with 100mM NaCL and 1%SDS with some tris and EDTA
> as well. Then I extract with an equal volume of
> phenol/chloroform/isoamyl alcohol, followed by an extraction with just
> chloroform. Then I add 1/10 volume of 3M NaAc, 2 volumes of 100% EtOH
> and centrifuge. Wash with 70% EtOH, dry, and resuspend in 10mM tris.
>

I digest the tail under pretty much the same conditions, but for
Southerns I do consecutive phenol and chloroform extractions (1 each)
and precipitate using an equal volume of isopropanol. Air dry the pellet
for a few minutes and dissolve in TE at 55C for as long as is necessary,
with shaking if possible, or by raking the tube over an Eppi rack. The
DNA will rarely fail to digest.