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.