Mini CTAB Extraction
1. Place sample in mortar containing liquid nitrogen and grind sample until a very fine
powder.
2. Transfer powder using a weighing boat into an eppendorf until about two thirds
full.
3. Add 500uL CTAB, mix well and incubate at 65oC for 30 minutes, shake the tubes
every 10 minutes.
4. In the fume cupboard add 500uL CIA, mix, open cap to release pressure, then
replace cap.
5. Put tubes on shaking platform for 30 minutes at 300rpm.
6. Centrifuge at 13,000rpm for 10 minutes.
7. Carefully remove the top aqueous phase to a new eppendorf tube, add 500uL CIA
and shake for 5 minutes.
8. Centrifuge at 13,000rpm for 10 minutes.
9. Remove the top aqueous phase to new eppendorf and add an equal volume of
phenol/chloroform.
10. Centrifuge at 13,000rpm for 5 minutes.
11. Remove the aqueous phase to a new eppendorf and add 1mL of chilled
Isopropanol (Propan-2-ol), mix and incubate on ice for 5 minutes.
12. Centrifuge at 13,000rpm for 10 minutes. Decant the supernatant, leaving the pellet
at the bottom of the tube.
13. Invert the tube on paper towel and allow to drain for 5 minutes.
14. Resuspend the pellet in 500uL sterile water, mix by gently tapping the tube. When
the pellet is no longer visible add one tenth volume (50 ul) 3M NaOAc and 2 volumes
(1 000 ul) of ice-cold 100% ethanol.
15. Incubate at -20oC for 10 minutes. Centrifuge at 13 000rpm for 20 minutes.
16. Decant the supernatant and add 400uL ice-cold 70% ethanol. Centrifuge at 13 000
rpm for 5 minutes.
17. Decant the supernatant and place tube in speed vac for 10 minutes.
18. Resuspend the pellets in 25-100uL TE+RNase and store at 4oC or sterile H20 and
freeze at -20oC.