DNA Isolation from whole blood

Robin Beech robin_beech at maclan.mcgill.ca
Thu Feb 1 08:50:30 EST 1996


In article <4el984$p7v at oracle.rz.uni-ulm.de>,
sabine.hanelt at medizin.uni-ulm.de (Sabine Hanelt) wrote:

> Hey netters,
> I am looking for an easy anf fast method for isolation of genomic DNA from 
> whole blood. I would like to isolate first the nuclei and then isolate DNA 

I was looking for the same thing about 2 months ago and came across this
protocol on a web site somewhere in France I think - can't remember where
exactly :-(

Hope it helps

Robin Beech,
  Institute of Parasitology,
  McGill University
  robin_beech at maclan.mcgill.ca

============================================================================
DNA Extraction from Whole Blood

Reference : Bell et al. (l981) PNAS 78: 5879-5583

The directions given here refer to 7 ml of whole blood. Blood should be
drawn using EDTA as an anticoagulant; heparin should be avoided as it is
an inhibitor of restriction enzymes. Blood may be stored for a few days at
room temperature, but is best stored at 4oC. High yields have been
obtained from blood refrigerated as long at 1 month. 

Isolation of crude WBC nuclei:

      1) Centrifuge blood sample in 50 ml conical disposable tube at 2000
rpm for 15 min at 0-4oC. Carefully remove most of the supernatant plasma
and discard. Take care not to discard the buffy coat which contains most
of WBC. 

      2) Resuspend the packed cells by adding 1X ice-cold BCL buffer (plus
triton) to a total volume of about 25 ml and vortex until the bottom of
tube is clean. Centrifuge 20 min at 2000 rpm at 0-4oC, discard supernatant
and drain pellet briefly.

      3) Resuspend pellet in 15 ml ice cold BCL buffer and vortex until
bottom of tube is clean. Centrifuge 15 min 2000 rpm at 0-4oC. Discard
supernatant an drain pellet in the cold for 5-10 min.. Pellet, which is
mostly white consists mainly of WBC nuclei.

      4) At this point you may freeze nuclei pellet at -70oC after
resuspending in 1.0 ml NL buffer containing 10% glycerol.
Isolation of DNA from WBC nuclei:

      5) Resuspend nuclear pellet in 2.5 ml ice cold NL buffer and keep on
ice. (If using frozen nuclei, thaw rapidly in 37oC bath, ice, and add 1.4
ml additional NL buffer to bring total volume of suspension to 2.5 ml)

      6) To another 2.4 ml portion of NL buffer at room temperature in a
15 ml polypropylene centrifuge tube, add 0.5 mg proteinase K, and 0.25 ml
10% SDS. 

      7) While gently vortexing the tube prepared in step 2, above, drip
the nuclear suspension into the proteinase K/SDS solution. This will lyse
the nuclei and should result in a noticeable increase in viscosity. The
floating crud which may appear would ultimately dissolve. Incubate 12-24
hours at 37oC with gentle rotation or shaking.

      8) Extract the preparation by shaking gently with an equal volume of
phenol / chlorofom / isoamy alcohol for 30 min. at room temperature.
Centrifuge 3000 rpm 10 min (Sorvall RT-6000. With a wide-bore plastic
pasteur pipette, transfer upper aqueous phase to a clean polypropylene
tube (At this point it is not necessary to be extremely careful to avoid
transferring interface material).

      9) Extract as in step 4, except using chloroform/isoamyl alcohol
(25:1) v/v instead of phenol.chloroform. Spin briefly in clinical
centrifuge; transfer upper aqueous phase to a clean 50 ml conical tube,
being careful to avoid contamination by interface crud or organic phase.

      10)   PPT. as usual with ammonium acetate at room temp, centrifuge
at 3000 rpm 5 min. Drain pellet.

      11)   Redissolve pellet overnight in 2.5 ml 0.1XTE at 0-4 deg with
gentle shaking. When dissolved, ppt with 7.5 m ammonium acetate as usual
(Room temp). V=Wash pellet with 70% ethanol and drain.

      12)   redissolve pellet in 1.0 ml TE with gentle shaking for 24
hours at 0-4oC.

Blood cell lysis buffer (5X BCL): 
      0.32 M sucrose, 
      10mM Tris-Cl-, ph7.5, 
      5mM MgCl2, 
      1% (v/v) tritonX-100. 

This is made as a 5X concentrate without triton, and autoclaved. Before
use, dilute 1:5 with sterile water and add 0.05 vols 20% (V/V) triton
X-100. 20%(v/v) is made using sterile water but is not autoclaved. 

Nuclear lysis buffer (NL): Autoclave and store at room temperature.
      75mM NaCl, 
      24mM EDTA, Ph8.0.



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