DNA isolation procedure

RPSCOTT RSCOTT at IRRI.CGNET.COM
Tue Aug 17 06:11:19 EST 1993


 
> > 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 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.

 Note:
 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-
 laced gels.


 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!

 R. Scott
 Plant Pathology Division
 International Rice Research Institute
 PO Box 933, 1099 Manila, Philippines
 e-mail: rscott at irri.cgnet.com


 
 
 



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