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 3 M sodium acetate and 2 volumes 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, 0.7 M 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.