Methods Digest, Vol 29, Issue 13

Analia Alet via methods%40net.bio.net (by analia_alet from intech.gov.ar)
Thu Oct 18 17:23:35 EST 2007


Hi, well here is a protocol we use with plant genomic DNA after the DNA 
extraction with CTAB. We use this protocol with plants that have high 
polysaccharide concentration

DNA + 0.5 vol NH4AC 7.5M
15 min in ice
centrifuge 30min 12000 rpm 4°C
SN + 2.5 vol absolute ethanol cold
place in ice
centrifuge 10 min 10000rpm
wash precipitate with ethanol 75% cold
centrifuge 10min 12000 rpm
dry leaving the tube upside down
Disolve in water. Add RNAse if needed


Hope it works
good luck

Lic. Analía I. Alet

Mensaje citado por methods-request from oat.bio.indiana.edu:

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.

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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