reply to ctab help
Thu Feb 17 00:03:06 EST 1994
In article <ydegefu.12.2D579347 at viikki21.helsinki.fi>, you wrote:
> Can anyone indicate a protocol for the use of CTAB in DNA purification? I
> will be grateful if you also add your experience in using this reagent.
> Thank you.
i have used CTAB for isolation of DNA from plants. and the DNA i used and
amt i estimated by reading the O.D at 260,280,300 &240 & RE digestions with
various enzymes indicated that the preparation was v.good.it is the
protocol of thomson et.al. with slight modifications:
extraction buffer (1X):
1%CTAB, 0.7M NaCl,50Mm Tris pH 7.5,EDTA 10mM pH 8, 0.1% §-Me.(optional if
polyphenols present then can use 100 mM Na-metabisulfite,5mM thiourea) 1%
Grind the tissue in liq. nitrogen. let it thaw a little. add extraction
buffer(5ml per gram tissue preheated at 65 degrees) incubate at 65 deg. for
15 min. let it come to RT. add equal amt. of chloroform:iAA(24:1).
centrifuge at 4000rpm (ss34 rotor). collect the supernatent and add one
tenth vol. of 10% CTAB containing 0.7 M NaCl. mix it properly and repeat
chloroform:iAA step.to the supernatent add equal amt of CTAB precipitation
buffer ( 1% CTAB, 50mMTris pH 8,10mM EDTA).mix & incubate for 30' at RT (if
spoolable DNA then spool it ) and centrifuge at 10000rpm for 10 min.
collect the ppt. dissolve it in high salt TE(1M NaCl+10 mMTris pH8+1mM EDTA
pH8).centrifuge to remove undissolved stuff. ppt the dna with 2.5 vols of
ethanol.wash the dna several times with 70% EtOH.dry & dissolve in Tris
(10mM)pH8 + 1mMEDTA.very rarely there is RNA contamination. i am sure the
dna thus obtained is of a v.good quality (don't compare with CsCl purified
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