Arabidopsis RNA miniprep
Adapted from Verwoerd et al, NAR 17:2362 (1989)
100 ug tissue scale
1. Collect leaf sample (1 leaf is enough if the plant is quarter-sized or larger) at 3-4 weeks after planting. Place tissue into labelled eppendorf tube, then add liquid nitrogen to flash freeze sample. Grind using drill with plastic pestle. Keep ground samples on dry ice until ready to extract or store at -20o C.
2. Add to frozen sample: 500 uL 80oC mixture of (water-saturated) phenol to extraction buffer (1:1 by volume); vortex to mix thoroughly (30 sec).
3. Add 250 uL chloroform, vortex.
4. Spin 5 minutes or more.
5. Save aqueous (top) phase; add 1/10th volume of 3.0 M NaOAc, then add 2 volumes EtOH, and place on ice or at -20oC for approx 30 minutes.
6. Spin 10 minutes in microfuge to pellet RNA, dry pellet.
7a. Resuspend in water (for 1 leaf use 5 uL).
7b. (Optional) To clean further: resuspend in 320 uL water, add 200 uL 8 M LiCl, set at -20oC several hours or 5oC overnight. Spin down, wash with 80% EtOH. Resuspend in water. Scale down, if a small amount of tissue is prepared. This optional step removes some of the DNA contamination, but can cause loss of some of the RNA yield, especially in small scale preps.
8. To run on gel, add 19 uL loading mix to RNA sample (in 5 uL water), heat to 65o C 10 minutes, load to gel. Run 1-5 ug RNA per lane. Electrophoresis is performed in 1X MSE buffer at 4-6 volts/cm until bromophenol blue migrates 1-3 cm. (e.g. 20 cm long x 13 cm gel: 120 volts). Photograph gel under UV.
To blot for northern blot analysis:
9. After photographing gel, capillary transfer RNA to nylon membrane in 6X SSC. UV-crosslink RNA to membrane, and hybridize according to Church and Gilbert (1984, PNAS 81: 1991).
Extraction Buffer 100 mM LiCl |
10 x MSE Buffer (1 L) 200 mM MOPS (41.86 g free acid) |
Formamide/BPB/XC 10 mg bromophenol blue
|
RNA Gel (300 mL) 260 ml water |
1 sample | 20 samples | |
Formamide/BPB/XC | 12.5 uL | 250 uL |
Formaldehyde | 4.25 uL | 85 uL |
10 X MSE Buffer | 2.5 uL | 50 uL |
EtBr (10 mg/mL) | 0.1 uL | 2 uL |