Litt-DNA Extraction from Whole Blood

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

The directions given here refer to 7 ml of whole blood. If different volumes are handled, scale up or down as appropriate.

All solutions, pipets, and glassware must be for eukaryotic work only. This precaution is necessary to avoid contamination of samples with prokaryotic DNA, a few pg of which can be detected on Southern blots.

Buffers and reagents:

1.) 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.

Recipe for 1 liter of 5X BCL buffer (-triton).

Sucrose 547 gr

2M Tris-Cl-, pH 7.5 25 ml

1M MgCl2 25 ml

Add deionized water to a final vol of 1 liter, filter sterilize,and store at room temp.

2.) Nuclear lysis buffer (NL): 75mM NaCl, 24mM EDTA, Ph8.0. Autoclave and store at room temperature (25 ml 5M NaCl, 48 ml 0.5 M EDTA to l liter.

3.) Phenol. Make sure you have some before your start. Equilibrated.

Isolation of crude WBC nuclei:

1.) Blood should be drawn using ACD or EDTA as 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 4 deg. C. High yields have been obtained from blood refrigerated as long at 1 month.

2.) Centrifuge blood sample in 50 ml conical disposable tube at 2000 rpm (5 on sorvall) for 15 min at 0-4 deg.. Carefully remove most of the supernatant plasma and discard. Take care not to discard the buffy coat which contains most of WBC.

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

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

5.) At this point you may freeze nuclei pellet at -70 deg. C after resuspending in 1.0 ml NL buffer containing 10% glycerol.

Isolation of DNA from WBC nuclei:

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

2.) 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.

3). 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 37 deg.. with gentle rotation or shaking.

4.) 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).

5.) 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.

6.) PPT. as usual with NaAcetate at room temp, centrifuge imediately at 3000 rpm 5 min. (not sure if immediately part is necessary). Drain pellet.

7.) 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.

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

9.) Read spectrum using 1: 20 dilution.

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