Dark treatment is crucial for reducing carbohydrate, polysaccharide, and polyphenolic compound production. For species that typically produce high amounts of these compounds, e.g., strawberry, 48 h is recommended. For high quality gDNA, tissue should be harvested and stored at − 80 °C. Storage times longer than 1 year, may result in degraded gDNA. For some species with low nuclei yield, or with low contents of the polyphenolic compounds, polysaccharides, tannins, and other secondary metabolites, for example, Arabidopsis, an increased amount of tissue is recommended, e.g., 20–40 g should be used in this protocol. For fiber-rich species, for example, maize, grinding the tissue to a fine powder is key.
The tissue must be mixed well in the flask very quickly; if the frozen tissue clumps, break it apart with a spatula or glass rod.
Miracloth should be outside (attached to the funnel) and cheesecloth be inside. Filtration step should not take more than 5 min.
The supernatant (or waste solution) containing BME must be stored as hazard waste in a fume hood.
Resuspend the pellets very carefully with a paint brush. The paint brush can be sterilized with 70% ETOH (make sure it is dry with no residual ethanol) or by UV-nicking.
The resuspension solution showing green color, suggests the chloroplasts are not completely disrupted by NIB. In this case, continue to repeat the wash, pellet and resuspend several times to remove the unwanted plastid DNA.
The final volume of the nuclei resuspension should not more than 2-ml.
20 ml of 2 × CTAB must be poured into the nuclei suspension very quickly, and immediately mix well by vortexing for 1–2 s. During incubation, invert the tube several times gently.
The chloroform extraction should be executed quickly within ~ 5 min.
When pipetting nuclei solution from organic extraction, using cut (large orifice) 5-ml tip.
Volume ratio of the 10% CTAB versus the supernatant should be 1:10.
During the last organic extraction, take caution not to disturb the interphase. Do not try to transfer the entire aqueous phase. If the interphase was transferred, spin @RT for another 10 min, then transfer the aqueous phase into a new tube).
When adding the 1 × CTAB precipitation buffer, and after mixing around 10 times by slow inversion, you should see the silk-like gDNA precipitate. When this becomes evident, do not continue to invert. This will aggregate the gDNA which will become too dense to dissolve.
If the pellet is difficult to dissolve, incubate at 56 °C for 5–10 min, and then incubate at 4 °C overnight in a refrigerator.
If there is a visible undissolved slurry before adding ethanol, spin at 3000×g for 5 min, then transfer the liquid phase into a new 2-ml tube for DNA precipitation.
Do not let the pellet become too dry. If the pellet is difficult to rehydrate, incubate at 4 °C overnight.
2-Mercaptoethanol (BME, Fisher Scientific)
Ethidium bromide (Fisher Scientific)
Midrange Lambda ladder PFG marker (NEB)
Lambda ladder PFG marker (NEB)
Yeast chromosome PFG marker (NEB)
Na2 EDTA (VWR)
Spermidine trihydrochloride (Sigma-Aldrich)
Spermine tetrahydrochloride (Sigma-Aldrich)
Sucrose, molecular biology grade (VWR)
Triton X-100 (Fisher Scientific)
Trizma base (Fisher Scientific)
Cyltrimethylammonium bromide, CTAB (MP Biomedicals)
Polyvinylpyrrolidone, PVP (MV: 40,000) (Sigma-Aldrich)
Equipment and supplies
Mortar and pestle, 0.5–1 L in volume (CoorsTek)
50 ml Conical tube (VWR)
Cheesecloth (VWR), cut into 25 × 25 cm2 pieces
Miracloth (Calbiochem, CAT# 475,855), cut Miracloth into 25 × 25 cm2 pieces
Funnel, 15 cm in diameter
Glass flask, 500 and 1000 ml
Magnetic stir bar
Paint brushes (12 Kid Art Brushes, Pinceaux d’artiste), sterilize by UV
Pipette tips, 200 μl, 1000 μl, 5000 μl
Swinging bucket centrifuge (Allegra X-15R, Beckman)
Water bath (Precision)
Pulsed-field gel electrophoresis (PFGE) systems: the CHEF-DR III System, Cat # 170-3695, 170-3700, and other parts (from 170-3690 to 170-3703, Bio-Rad)
HB (homogenization buffer) stock (10×)
For 10 × stock (100 mM Trizma base, 800 mM KCl, 100 mM EDTA),
12.1 g of Trizma base,
59.6 g of KCl,
37.2 g of Na2 EDTA,
to ~ 800 ml of ddH2O in a 1-l PYREX bottle. Stir until dissolved. Adjust the pH of the solution to 9.2 with NaOH and bring to a final volume of 1000 ml in a 1000-ml graduated cylinder. Transfer the solution to a glass stock bottle and store it at 4 °C. The 10 × HB stock may be stored at 4 °C for up to 1 year.
HB solution (1×)
To prepare stock solution (1 × HB, 0.5 M sucrose, 1 mM spermidine trihydrochloride, 1 mM spermine tetrahydrochloride)
100 ml of 10 × HB stock
171.2 g of sucrose to ~ 700 ml of ddH2O
0.255 g of spermidine trihydrochloride
0.348 g of spermine tetrahydrochloride
in a 1-l PYREX bottle. Stir until dissolved. Bring to a final volume of 1000 ml in a 1000-ml graduated cylinder. Transfer the solution to a glass stock bottle and store it at 4 °C for up to 3 months.
Triton X-100 (20% (vol/vol) Buffer
For 20% (vol/vol) Triton X-100 buffer [1 × HB, 0.5 M sucrose, 20% (vol/vol) Triton X-100],
20 ml of Triton X-100
10 ml of 10 × HB stock
17.15 g of sucrose to ~ 60 ml of ddH2O in a 100-ml beaker.
Stir until dissolved. Bring to a final volume of 100 ml in a 100-ml graduated cylinder.
Transfer the solution to a glass stock bottle and store it at 4 °C. The 20% Triton X-100 stock may be stored at 4 °C for 1 year or longer without major problems.
NIB buffer (Nuclei Isolation Buffer)
Make the buffer (1 × HB solution, 0.5% Triton X-100, 0.5% (vol/vol) 2-Mercaptoethanol) just before use. The volume of the buffer needed is 10–15 ml per gram weight of the sample, including nuclei isolation and sub-sequent washes.
For 200 ml of the buffer,
195 ml of 1 × HB
5 ml of 20% (vol/vol) Triton X-100 buffer
Mix well and keep it at 4 °C or on ice.
Immediately before use, add 1 ml of BME to the buffer.
Always use freshly made buffer for megabase-sized DNA isolation
2 × CTAB buffer (pH = 9.2)
2% CTAB (w/v)
100 mM Tris
20 mM EDTA (ethylenediaminetetraacetic acid)
1.4 M NaCl
1% PVP (polyvinylpyrrolidone) Mr 40000
10% × CTAB buffer
0.7 M NaCl
1 × CTAB precipitation buffer (pH = 9.2)
50 mM Tris
10 mM EDTA
High salt TE buffer (pH = 9.0)
10 mM Tris
1 mM EDTA
1 M NaCl
0.1 × TE buffer (pH = 9.0)
1.0 mM Tris
0.1 mM EDTA