H. Genomic DNA isolation from blood

 

Genomic DNA isolation is performed according to the FBI protocol (27). After the blood samples (stored at -70degC in EDTA vacutainer tubes ) are thawed, standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55deg C for one hour. The sample then is phenol extracted once with a phenol/chloroform/isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer, and then ethanol precipitated a second time. Once the pellet is dried, buffer is added and the DNA is resuspended by incubation at 55degC overnight, the genomic DNA solution is assayed by the polymerase chain reaction.

Protocol   (updated 10/30/98)

1. Blood samples typically were obtained as 1 ml of whole blood stored in EDTA vacutainer tubes frozen at -70deg C.

2. Thaw the frozen samples, and to each 1 ml sample, add 0.8 ml 1X SSC buffer, and mix. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

3. Remove 1 ml of the supernatant and discard into disinfectant.

4. Add 1 ml of 1X SSC buffer, vortex, centrifuge as above for 1 minute, and remove all of the supernatant.

5. Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 ul of 10% SDS and 5 ul of proteinase K (20 mg/ml H2O) (Sigma P-0390), vortex briefly and incubate for 1 hour at 55degC.

6. Add 120 ul phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.

7. Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of cold 100% ethanol, mix, and incubate for 15 minutes at -20deg C.

8. Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant and drain.

9. Add 180 ul 10:1 TE buffer, vortex, and incubate at 55degC for 10 minutes.

10. Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

11. Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute at 12,000 rpm in a microcentrifuge.

12. Decant the supernatant, and dry the pellet in a Speedy-Vac for 10 minutes (or until dry).

13. Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at 55degC, vortexing periodically to dissolve the genomic DNA. Store the samples at -20degC.