non organic dna extraction
danielg at uncmed.med.unc.edu
danielg at uncmed.med.unc.edu
Mon Feb 19 08:34:23 EST 1990
Hi friends. I had many requests concerning my posting on non-organic
extraction of DNA, so will now post my main reference. For those of you
who wrote me and have not received a personal response, I tried but the
postman won't deliver. Please excuse the typo's. Reference for the
following is Nucleic Acids Research Vol 16 No 3 1988, p. 1215.
One of the obstacles encountered when extracting DNA from a large
number of samples is the cumbersome method of deproteinizing cell
digests with the hazardous organic solvents phenol and
isochloroform. Several other non-toxic extraction procedures have
been published, but require either expensive dialysis (1) or the
use of filters (2). A rapid, safe and inexpensive method was
developed to simplify the deproteinization procedure. This method
involves salting out of the cellular proteins by dehydration and
precipitation with a saturated NaCl solution.
Buffy coats of nucleated cells obtained from anticoagulated blood
(ACD or EDTA) were resuspended in 15 mL pp centrifue tubes with 3
mL of nuclei lysis buffer ( 10 mM Tris-HCl, 400 mM NaCl and 2 mM
Na2EDTA, ph 8.2). The cell lysates were digested overnight at 37
degrees with 0.2 mL of 10% SDS and 0.5 mL of a protease K solution
(1 mg protease-K in 1% SDS and 2 mM Na2EDTA).
After digestion was complete, 1 mL of saturated NaCl
(approximately 6 M) was added to each tube and shaken vigourously
for 15 seconds ( my note: will foam), followed by centrifugation
at 2500 rpm for 15 minutes. The precipitated protein pellet was
left at the bottom of the tube and the supernatant containing the
DNA was transferred to another 15 mL pp tube. Exactly 2 volumes
of room temperature absolute ethanol was added and the tubes
inverted several times until the DNA precipitated. The
precipitated DNA strands were removed with a plastic spatula or
pipette and transferred to a 1.5 mL microcentrifuge tube
containing 100-200 uL TE buffer (10 mM Tris-HCl, 0.2 mM Na2EDTA,
ph 7.5). The DNA was allowed to dissolve 2 hours at 37 degrees C
The DNA obtained from this somple technique yielded quantities
comparable to those obtained from phenol-chloroform extractions.
The 260/280 ratios were consistently 1.8-2.0, demostrating good
deproteinization. Restrictions were performed using a number of
different enzymes requiring both high, medium or low salt
concentrations, all resulting in complete restriction. This
procedure has been used in our laboratory on several thousand
blood samples for parentage, population and forensic studies.
This technique is used with our nonisotopic hybridization
procedures (3) rendering the entire process of RFLP analysis free
of toxic materials
1. Longmire, J., Albright, K., Lewis, A., Meincke, L. and
Hildebrand, C. (1987) Nucleic Acids Res. 15, 859.
2. Leadon, S. and Cerutti, P. (1982) Anal. Biochem. 120, 282-8.
3. Dykes, D., Fondell, J., Watkins, P. and Polesky, H. (1986)
Electrophoresis 7, 278-282.
I have used this successfully on whole blood, as well as cell culture
(HL60 cells) and frozen tissues (lymph nodes and tonsil). Works great.
One poster asked me if it will work on fatty tissues, all I can say is try
it, I'm not sure, but the chloroform may have helped in the old method
(that's what we call p/c extraction around here!!).
The article noted that low salt restrictions work ok, but I have a friend
who has had some difficulty. He has worked around this by adding a 70%
ethanol wash after the ethanol precipitation step, and, if restriction of
the resuspended DNA does not work (rare), he reprecipitates the DNA, and
resuspends. Has never had any problem afterwards.
Daniel G. Sinclair
Research Tech II
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