DNA from blood

ROGER GREEN,MEDICINE,ST.JOHN'S,NF,CAN roger at kean.ucs.mun.ca
Tue Nov 21 08:07:50 EST 1995


> Does anyone have a good protocol, or reference to a protocol, for
> preparing genomic DNA from blood samples? Also, are there any commercial
> kits available for the same purpose that are worth the extra expense of a
> kit? If you have any helpful information please respond by e-mail if
> possible.


We have isolated DNA from thousands of blood samples using the following 
method.
______________________________________________________________________
Molecular Genetics Research, Faculty of Medicine,
Memorial University of Newfoundland
                                       
                                       
                        Preparing DNA from Whole Blood
                                       
                                       
                  Collect blood in EDTA (purple top) tubes.
                                       
                   Volumes are given for 5-7 mls of blood.
                                       
                                       
1.   Add 5 vols warm (37o) NH4Cl:Tris soln (900ml of 0.155 M
NH4Cl;100ml of 0.17 M  Tris.HCl, pH 7.65) to 1 vol blood in 50ml
cent. tube.  Incubate at 37o for 5 mins. 

2.   Cent. at 2500 rpm  5 mins (1000 x g).

3.   Pour off supernatant leaving white cell pellet at bottom.

4.   Add 10ml saline (0.85% NaCl).  Vortex and centrifuge again.

5.   Pour off supernatant. Add 3ml of nuclei lysis buffer (10mM Tris
HCl, 400mM NaCl, 2mM  EDTA, pH 8) to the cell pellet. Vortex
and transfer to 15 mL cent tube.

6.   Add 0.2 ml of 10% SDS and 0.5 ml of pronase E solution
(3mg/ml in 1% SDS, 2 mM EDTA).

7.   Incubate at 37o overnight (or 2hr at 55o or a day or two at room
temp).

8.   Add 1ml of saturated NaCl, shake vigorously for 15 secs and
centrifuge at 2500 rpm for 15  mins.

9.   Gently pour supernatant into a 15ml tube and add 2 vols of
absolute ethanol; invert the tube several times to ppt the DNA.

10.  Fish out DNA clump out with 9" glass hook.  (9" pasteur
pipette; tip melted to form a hook).  Wash several times with a
stream of 70% ethanol.  Let air dry.

11.  Dissolve DNA in 300-500 æl TE (10 mM Tris, 1 mM EDTA,
pH 8) overnight. Simply break the last 1 cm or so off the pasteur
pipet and put into 1.5 mL microtube with the TE. Gently mix sample
on a rotator for an hour or more before quantitating it by UV
spectrometry. The glass fragment can be left in the tube to aid
mixing.

     DNA sample can be stored at 4o or frozen for extended period

_____________________________________________________________________
Roger C. Green,	Faculty of Medicine               Phone: (709)737-6884
Memorial University , St. John's, Newfoundland    FAX  : (709)737-7010



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