> > Dinakar Desai at "DAC050 at marshall.wvnet.edu" (17-AUG-1993 00:25):
> > Is there any quick method to isolate DNA from cell lines for Southern blots
> > other than going through Proteinase K method.
Try the ff. procedure (in which organic solvent extraction is optional):
1. Macerate or pulverize the tissue in liquid nitrogen.
2. Put the ground tissue in a microfuge tube (about 200-250 ul vol).
3. Suspend the each ground tissue in 500 ul extraction buffer (100 mM TrisHCl,
pH 8; 100 mM EDTA; and 250 mM NaCl).
4. Add 50 ul 10% SDS. Vortex the mixture to ensure thorough suspension of
the sample and incubate at 65oC for 30 min. Flick the tube intermittently.
5. Add 200 ul 3M potassium acetate-2M acetic acid solution (pH 4.8). Flick the
tube lightly but make sure thorough mixing is provided. The KOAc solution
should precipitate SDS and substantial amounts of proteins.
6. Chill the samples over ice or put in a freezer for 10-15 min.
7. Centrifuge at about 12,000 rpm for 10 min and transfer 500-600 ul of the
supernatant carefully into a fresh tube containing 1 ml absolute ethanol.
Invert the tube several times to ensure complete nucleic acid pptn.
8. Pellet the nucleic acids by centrifugation for 5 min, discard the super-
natant and dry the pellet in a vacuum but not to complete dryness.
9. Resupend the pellet in 250 ul TE buffer containing 20 ng/ul DNAse-free
RNAse A and incubate for 1 h at 37oC.
10. Add 25 ul 3M KOAc-2M HOAc and 500-625 ul absolute ethanol. Mix the tube
contents gently but thoroughly to quantitatively precipitate the DNA.
11. Pellet the DNA by a 3-5 centrifugation, wash with 500 ul 70% ethanol,
air-dry, and resuspend in about 100-200 ul TE buffer.
If RNA contamination will not cause interference (cross-hybridization with
the probe) during Southern, forget step 9. Simply rinse the nucleic acid
pellet with 70% ethanol, dry, and dissolve in TE. Obviously, however,
you cannot quantify your DNA yield correctly by absorbance spectrophotometry.
Sound alternatives would be fluorometry and UV-visualization of ethidium-
The above procedure was modified from the procedure for yeast reported in
Current Protocols in Molecular Biology by D.A. Treco. In our hands the
modification without RNAse treatment consistently yields satisfactory-
quality rice blast fungal DNA which readily cut with most restriction enzymes.
But should you harvest DNA using our procedure which resists endonucleolytic
attack, try supplementing the procedure with a chloroform-isoamyl alcohol
extraction after the RNAse-treatment.
So, good luck!
Plant Pathology Division
International Rice Research Institute
PO Box 933, 1099 Manila, Philippines
e-mail: rscott at irri.cgnet.com