TITLE: CTAB Extraction from Sample Ground in Liquid Nitrogen
AUTHORS: Jordan Beasley

[DESCRIPTION]
This protocol describes the process of grinding a sample in liquid nitrogen and extracting DNA with a CTAB extraction.

[STEPS]
SECTION: Cryogenic Grinding and CTAB Extraction
1. Dispense some liquid nitrogen in a small dewer (small thermos size)
SECTION: Cryogenic Grinding and CTAB Extraction
2. Fill a mortar with liquid nitrogen to cool it and let it boil off. When it has completely boiled away, add a small amount more into the mortar and grind with pestle to a fine powder. Scrape the powder into a 1.5ml tube.
SECTION: Cryogenic Grinding and CTAB Extraction
3. Add 500µl CTAB, 50µl sarkosyl and 50µl Proteinase K to the powder and vortex to mix.
SECTION: Cryogenic Grinding and CTAB Extraction
4. Place tube in a hot block at 60°C overnight or if in a rush 60-90 minutes.
SECTION: Cryogenic Grinding and CTAB Extraction
5. After incubation add 300µl of phenol chloroform (This extracts the DNA into an aqueous layer).
SECTION: Cryogenic Grinding and CTAB Extraction
6. Mix by inversion for 15 minutes on a rotator if you need high molecular weight. If you don't need high molecular weight, vortex well.
SECTION: Cryogenic Grinding and CTAB Extraction
7. Centrifuge for 3 minutes at 16 rpm (max speed) and label a new tube.
SECTION: Cryogenic Grinding and CTAB Extraction
8. Carefully remove the top aqueous layer and transfer to the new tube. Avoid disturbing any of the white protein layer.
SECTION: Cryogenic Grinding and CTAB Extraction
9. Add 500µl SEVAC and mix by placing on a rotator for 15 minutes (or vortex well if you don't require high molecular weight DNA).
SECTION: Cryogenic Grinding and CTAB Extraction
10. Centrifuge for 3 minutes at 16 rpm (max speed) and label a new tube.
SECTION: Cryogenic Grinding and CTAB Extraction
11. Carefully remove the top aqueous layer and transfer to the new tube. Avoid disturbing any of the white protein layer.
SECTION: Cryogenic Grinding and CTAB Extraction
12. Repeat steps 9-11.
SECTION: Cryogenic Grinding and CTAB Extraction
13. Add 400µl isopropanol and mix by inverting - DO NOT VORTEX! You may see DNA precipitate out.
SECTION: Cryogenic Grinding and CTAB Extraction
14. Centrifuge for 3 minutes at 16 rpm (max speed), you might see a pellet.
SECTION: Cryogenic Grinding and CTAB Extraction
15. Remove as much of the liquid as you can without disturbing the pellet.
SECTION: Cryogenic Grinding and CTAB Extraction
16. Add 500µl 70% ethanol (to wash away isopropanol)
SECTION: Cryogenic Grinding and CTAB Extraction
17. Wash pellet by inverting several times
SECTION: Cryogenic Grinding and CTAB Extraction
18. Centrifuge for 3 minutes at 16 rpm (max speed) and remove as much ethanol as possible without disturbing the pellet.
SECTION: Cryogenic Grinding and CTAB Extraction
19. Place tube on a hot block with the lid open at 60°C for 5 minutes to evaporate off any remaining ethanol.
SECTION: Cryogenic Grinding and CTAB Extraction
20. Resuspend in 30-50µl sterile water (flick the tube to help the pellet dissolve and if stubborn, pop back on the hot block for a few minutes).
SECTION: Cryogenic Grinding and CTAB Extraction
21. Quantify extract on Nanodrop and/or Qubit.