- Add Diethyl Dithiocarbamic Acid Sodium salt (4 mg/ml) and RNAase A (100 ug/ml) to an aliquot of Grinding Buffer, and B-mercaptoethanol (1%) to an aliquot of Lysis Buffer (see below).
- Use a single newly emerged, still rolled-up leaf (~10 mg, the newer the better). Place foliage in microfuge tubes in a liquid nitrogen bath.
- Add approximately 200 to 400 ul of liquid nitrogen to a tube containing foliage. Grind approx. 10-15 seconds using a motorized pestle. Stop grinding when liquid nitrogen evaporates and tissue defrosts.
- Add 200 ul Grinding Buffer. Grind another 10 seconds until tissue is well-homogenized.
- Place tube in hot water bath (~40-65 deg.) and incubate while grinding other samples. Each sample should be incubated at least 10 min.
- Add 200 ul Lysis Buffer to each tube. Mix by inverting several times.
- After grinding all samples (typically as many as will fit in microfuge), incubate at 65 deg. for 30-60 min. Every 10 to 15 minutes, mix by inverting tubes several times.
- Add equal volume of phenol:chloroform:IAA (25:24:1). Mix well by inverting at least 10 times. Spin 8-10 min. at 12,000 rpm at room temperature.
- Remove 50-70% of supernatant to new tube, taking care to avoid interface. If supernatant is cloudy or contaminated with interface, go to step 10. Otherwise skip to 12.
- (Optional) Add an equal volume of chloroform, mix well by inverting at least 10 times. Spin 5 min. at 12,000 rpm at room temperature.
- Remove 50-70% of supernatant to new tube, taking care to avoid interface.
- Add 2/3 volume of isopropanol. Mix by inverting.
- Incubate 15-30 min. on ice or room temperature.
- Spin in microfuge 5-10 minutes at room temperature.
- Decant supernatant and blot tubes on kimwipe.
- Dry in speed-vac for 2-4 minutes until no isopropanol odor is apparent.
- Add 50 ul TE buffer (10:1), pH 8. Let sit at room temperature for 10-20 min., or at 4 deg. overnight (preferred). Mix by flicking tube several times.
100 mM Tris pH 8
20 mM EDTA pH 8
4 mg/ml diethyl dithiocarbamic acid, sodium salt, added just prior to use.
100 ug/ml RNAase A, added just prior to use
Lysis Buffer
100 mM Tris pH 8
20 mM EDTA pH 8
1 M NaCl
2% SDS
1% B-mecaptoethanol, added just prior to use
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