GENOMIC
DNA ISOLATION from WHOLE BLOOD
Protocol:
Blood samples will be taken in EDTA
vacutainer tubes. Fresh samples can be used immediately or stored at -70° C.
If frozen, thaw before use. Remove
one mL of blood and add 0.8 ml 1X SSC buffer, and mix. Centrifuge for one
minute at 12,000 rpm in a microcentrifuge.
Remove the supernatant and discard
into a disinfectant (e.g. Chlorox®).
Add 1 ml of 1X SSC buffer, vortex,
centrifuge as above for 1 minute, and remove all of the supernatant. Discard
the supernatant into a disinfectant.
Add 375m l
of 0.2M NaOAc to each pellet and vortex briefly. Then add 25m l
of 10% SDS and 5m l of proteinase K (20 mg/ml H2O)
(Sigma P-0390), vortex briefly and incubate for 1 hour at 55° C.
Add 120m l
phenol/chloroform/isoamyl alcohol (25:24:1) and vortex for 30 seconds.
Centrifuge the sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.
Carefully remove the aqueous layer
to a new 1.5ml microcentrifuge tube, add 1ml of cold 100% ethanol, mix, and
incubate for 15 minutes at -20° C.
Centrifuge for 2 minutes at 12,000
rpm in a microcentrifuge. Decant the supernatant and drain.
Add 180m l
TE buffer, vortex, and incubate at 55° C for 10 minutes.
Add 20m l
2M sodium acetate and mix. Add 500m l of cold 100% ethanol, mix, and centrifuge
for 1 minute at 12,000 rpm in a microcentrifuge.
Decant the supernatant and rinse
the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute at 12,000 rpm in a
microcentrifuge.
Decant the supernatant, and dry the
pellet in a Speed-Vac for 10 minutes (or until dry).
Resuspend the pellet by adding 200m l
of TE buffer. Incubate overnight at 55° C, vortexing periodically to dissolve the
genomic DNA. Store the samples at -20° C.