Genomic DNA extraction from Arabidopsis

1. Harvest 2-4 weeks arabidopsis plants and freeze with liquid Nitrogen and freezer-dried overnight.
2. Prior to grinding the tissue, active every 30ml of 2% CTAB with 150ul 2-merceotoethanol. Place it in a 60^oC water bath to heat.
3. Ground the freezer-dried tissue at room temperature, add actived CTAB (30ml for each 2-3 g tissue in fresh weight), transfer to a large centrifuge tube. Incubate at 60^oC for 30min, swirl occationally.
4. Add 50ml chloroform (1:1 v/v), mix gently but thoroughly.
5. Centrifuge at room temperature 5000rpm for 10min.
6. Take the aqueous phase (top) and filter through Miracloth (Calbiochem) into a clean tubes, then add 1vol of cold isopropanol and mix gently.
7. Centrifuge 3000rpm at 0^oC, 10 min., remove supernatant.
8. Add 30ml 75% ethanol (containing 10mM NH4OAc) to the pellet, 4^oC for few minuts to 2 days.
9. Centrifuge 8500rpm at 0^oC, 15 min. Remove wash buffer and allow to air dry for 5 min.
10. Resuspend in 5ml TE buffer, Stop if disired.
11. Add 5ul 10mg/ml RNase A, incubate 30 min. at 37^oC.
12. Add 500ul (1/10 Vol) 3M NaAc (pH7.7) and 5ml phenol/chloroform, mix gently.
13. Spin at 8000rpm at R.T. for 5 Min.
14. Collect the top phase and transfer to a new tube, precipitate DNA with 2.5 Vol cold ethanol.
15. Centrifuge 10,000rpm for 10 min. at 4^oC, remove supernatant and airdry.
16. Dissolve in TE buffer (500ul to 1000ul).

• 1.4M NaCl
• 2% CTAB
• 20 mM EDTA
• 100 mM Tris-HCl (pH8).

• 0.2% 2-mercaptoethanol was added before use