4.1　 Genomic DNA extraction

Naoki Aono, Yoshikatsu Sato and Tomoaki Nishiyama

(1) CTAB method

This method is for the extraction of pure genomic DNA in large scale.

Procedure

1. Blend 8~15 mm size colony by polytron or glass beads and spread onto 3~4 plates with solid BCDATG medium laid with cellophane. Culture for 8-10 days at 25˚C under continuous light condition.

2. Harvest the protonemal cells. It is important to harvest the cells before they turned to brown.

3. Remove excess water by wrapping with paper towel.

4. Measure and note the fresh weight of the tissue.

5. Grind the tissue with mortar and pestle under liquid nitrogen.

6. Transfer the cell powder in a 50 ml conical tube and store in –80˚C freezer to evaporate nitrogen.

7. Heat 2x CTAB buffer with microwave oven just before boiling.

8. Add 4 ml of hot 2x CTAB buffer to the cell powder and thaw the powder with gently mixing at 60˚C.

9. Incubate the suspension at 60˚C for 1 hour in water bath with gently shaking.

10. Add 5 ml of Chloroform:Isoamylalcohol. Mix using rotator for 30 min.

11. Spin at 4000 rpm for 20 min (Hitachi himac CR20E, rotar: R12A2). Transfer the aqueous phase to 10 ml centrifuge tube.

12. Add 0.4 ml of 10% CTAB to the supernatant.

13. Extract the solution with 5 ml of Chloroform:Isoamylalcohol twice (rotar: RPR20-3-334).

14. Transfer the last aqueous phase to 40 ml centrifuge tube.

15. Add an equal volume of CTAB ppt buffer.

16. Spin at 6000 rpm for 20 min (rotar: RPR20-2-621). Discard the supernatant.

17. Dissolve the pellet in 5 ml of NaCl/TE at 60˚C.

18. Add 2 volume of ethanol.

19. Spin at 18000 rpm for 10 min. at 25˚C. Discard the supernatant.

20. Rinse the pellet with 70% ethanol.

21. Dissolve the pellet in 400 µl of TE. Transfer to 1.5 ml tube.

22. Add 1 µl of 2 mg/ml RNaseA and incubate at 37˚C for 30 min.

23. Store on ice for a while.

24. Extract the solution with an equal volume of TE-saturated phenol.

25. Extract the solution with an equal volume of chloroform.

26. Spin at 15000 rpm for 10 min at 4˚C. Transfer the supernatant to new tube.

27. Add 1/10 volume of 3M sodium acetate and 2 volume of ethanol.

28. Spin at 15000 rpm for 10 min. Discard the supernatant.

29. Dissolve the pellet in 400 µl of TE.

30. Add 0.6 volume of PEG solution and store on ice for 30 min.

31. Spin at 15000 rpm for 10 min at 4°C. Discard the supernatant.

32. Dissolve the pellet in 400 μl of TE.

33. Add 40 µl of 3 M sodium acetate and 1 ml of ethanol.

34. Spin at 15000 rpm for 10 min. Discard the supernatant.

35. Rinse the pellet with 500 µl of 70% ethanol. Discard the supernatant.

36. Dissolve the pellet in 200 µl of TE.

Solutions required

Liquid nitrogen

2x CTAB buffer

2% CTAB, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0, 20 mM EDTA

Chloroform:Isoamylalcohol

Chloroform:Isoamylalcohol　 25:1

10% CTAB

10% CTAB, 0.7M NaCl

CTAB ppt buffer

1% CTAB, 50 mM Tris-HCl pH 8.0, 10 mM EDTA

NaCl/TE

1 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA

PEG solution

2M NaCl, 20% PEG8000

Ethanol

70% Ethanol

TE-saturated phenol

Chloroform

3 M sodium acetate pH 7.0

2 mg/ml RNaseA

Note

15 mg ~ 25 mg DNA/g fresh weight is usually obtained.

(2) Simplified CTAB method

This method is suitable for the extraction of low grade genomic DNA in a small scale for such as PCR screening. Young green colony and young gametophores can be used for DNA preparation.

Procedure

1. Place the cells into 1.5 ml tube containing 400 µl of 2x CTAB buffer.

2. Grind the sample with pestle or pipette tip.

3. Incubate at 60˚C in water bath for 1 hour.

4. Extract the solution with an equal volume of Chloroform:Isoamylalcohol.

5. Spin at 15000 rpm for 10 min.　 Transfer the aqueous phase to new tube.

6. Add an equal volume of 2-propanol.

7. Spin at 15000 rpm for 10 min.　 Discard the supernatant.

8. Rinse the pellet with 70% ethanol.

9. Spin at 15000 rpm for 5 min.　 Discard the supernatant.

10. Dissolve the pellet in 50 µl of TE containing 1 ml of 1 mg/ml RNaseA

Solutions required

2x CTAB buffer

2% CTAB, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0, 20 mM EDTA

Chloroform:Isoamylalcohol

Chloroform:Isoamylalcohol　 25:1

2-propanol

70% Ethanol

1 mg/ml RNaseA

Note

We use 0.5 µl ~ 1.0 µl as a PCR template (total 20 µl PCR volume).

(3) Automatic genomic DNA isolation by KURABO NA-2000

Genomic DNA isolated by automatic nucleic acid isolation system (KURABO NA-2000) is available for genomic Southern blotting as well as genomic PCR.　

Materials

1. Protonemal cells cultured under white light for 5-7 days in BCDATG medium.

Or gametophyte colony cultured under white light for 2-4 weeks (colony size: 1-2 cm in diameters) in BCDAT medium covered with a cellophane membrane.

2. 6-well tubes (PT5000, KURABO) as a sample tube and a collection tube

Procedure

1. Fresh Samples (DO NOT FREEZE) in the sample tube were pounded up by crushing equipment (KURABO SH-48). The condition is 1200 rpm for 2 minutes.

2. The sample tube was placed into liquid nitrogen.

3. Pound up again at 1200 rpm for 2 minutes.

4. Add 0.43 ml Plant cell lysis solution (NO. 7) in each well and incubate for 1 h at 65˚C.

5. Set sample tubes and collection tubes at NA-2000 and start program Ver. 1.

6. After the program is finished, collection tubes should be dried up for overnight.

7. Add 100 µl TE with 100 µg/ml RNaseA in each well and incubate for 1 h at 37˚C.

8. Purify DNA by PEG precipitation.

9. Dissolve with 100 µl TE.