Conifer RNA prep: Difference between revisions
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''Modified Le Provost Extraction buffer* (2% CTAB, 1% polyvinylpyrrolidone K-60, 100mM Tris-HCl (pH 8.0), 25mM EDTA, 2.0 M NaCl, 3.5 mM spermidine free acid HRS (add before use, 0.69M [0.1 g/mL] stock), 20 mM DTT (add before use, 1M stock). Modified from Le Provost et al., 2007, Biol. Res. 40:291-297'' | ''Modified Le Provost Extraction buffer* (2% CTAB, 1% polyvinylpyrrolidone K-60, 100mM Tris-HCl (pH 8.0), 25mM EDTA, 2.0 M NaCl, 3.5 mM spermidine free acid HRS (add before use, 0.69M [0.1 g/mL] stock), 20 mM DTT (add before use, 1M stock). Modified from Le Provost et al., 2007, Biol. Res. 40:291-297'' | ||
'''Extraction buffer, 50 ml: Add in this order…''' | '''Extraction buffer, 50 ml: Add in this order…''' | ||
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• 1.5 mL centrifuge tube (2 per sample) | • 1.5 mL centrifuge tube (2 per sample) | ||
'''Zymo supplies''' | |||
Zymo supplies | |||
• Zymo-spin IIC column (1), collection tube (1), 1.5mL tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added) | • Zymo-spin IIC column (1), collection tube (1), 1.5mL tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added) | ||
Reagents | '''Reagents and Materials''' | ||
• 5 M NaCl, DEPC Water, 22% PVP K-60, 0.1 g/mL spermidine, 1 M DTT, 100% EtOH, 70% EtOH, Glycoblue coprecipitant, RNA Storage Solution | • 5 M NaCl, DEPC Water, 22% PVP K-60, 0.1 g/mL spermidine, 1 M DTT, 100% EtOH, 70% EtOH, Glycoblue coprecipitant, RNA Storage Solution, Dry ice flakes or chips (about 5 lbs), Heat blocks set to 65°C | ||
Revision as of 00:21, 14 November 2010
Materials Required for Total RNA Extraction from Douglas-fir Needles
Modified Le Provost Extraction buffer* (2% CTAB, 1% polyvinylpyrrolidone K-60, 100mM Tris-HCl (pH 8.0), 25mM EDTA, 2.0 M NaCl, 3.5 mM spermidine free acid HRS (add before use, 0.69M [0.1 g/mL] stock), 20 mM DTT (add before use, 1M stock). Modified from Le Provost et al., 2007, Biol. Res. 40:291-297
Extraction buffer, 50 ml: Add in this order…
• 5 ml 1 M Tris-HCl, pH 8.0
• 2.5 mL 0.5 M EDTA, pH 8.0
• 20 mL 5 M NaCl
• 2.3 ml PVP K-60, 22%
• 1 gram CTAB
• add water up to 48.7 mL (about 18 mL), mix well (heating to 50°C may help)
---- then, just before use…
• 0.25 mL of 0.1 g/mL spermidine
• 1 mL of 1 M Dithiothreitol
CIA, 24:1 [V/V]):
• 48 ml chloroform
• 2 ml isoamyl alcohol
7.5 M LiCl: • Ambion RNA Precipitation Solution
Tubes: • Fast-Prep grinding tubes (1 per sample, 2 beads each) • 2 mL centrifuge tube (1 per sample) • 1.5 mL centrifuge tube (2 per sample)
Zymo supplies • Zymo-spin IIC column (1), collection tube (1), 1.5mL tube (1), RNA Lysis Buffer, RNA Prep Buffer, RNA Wash Buffer (with EtOH added)
Reagents and Materials • 5 M NaCl, DEPC Water, 22% PVP K-60, 0.1 g/mL spermidine, 1 M DTT, 100% EtOH, 70% EtOH, Glycoblue coprecipitant, RNA Storage Solution, Dry ice flakes or chips (about 5 lbs), Heat blocks set to 65°C
Detailed Notes for Total RNA Extraction from Douglas-fir Needles
Day 1
1. Samples (100 mg-200 mg) should be placed in a Fast-Prep grinding tube containing two ceramic beads (with or without garnet – doesn’t matter)
2. Add flaked dry ice to the 24 position Fast-Prep teflon grind adaptor. Grind tissues using two pulses of 20 seconds each, angular momentum of 4. Make sure that the tissue stays frozen and that it is ground to a fine powder.
3. After grinding, add 1 mL of hot (65C) RNA Extraction Buffer to each tube. Shake or vortex vigorously until the ground dried tissue is completely resuspended.
4. Incubate mixture for 15 min in a heat block at 65C; mix vigorously by inverting tubes every 3 min.
5. Place the tubes in the centrifuge; spin at 9500g x 10 min, room temperature.
6. Transfer supernatant (~850-900 l) to a clean 2 mL RNase-free tube. Add 1 volume of 24:1 CIA to each sample, mix well by inverting tube for 30 seconds.
7. Place the tubes in the centrifuge; spin at 9500g x 5 min, room temperature.
8. Transfer supernatant (~600-700 l) to a clean 1.5 mL RNase-free tube.
--- optional back-extraction for maximal RNA recovery --- 8b. Add 100l of RNA Extraction Buffer to the aqueous layer of the sample tube containing chloroform. 8c. Mix well, then re-centrifuge at 9500g x 5 min, room temperature. 8d. Remove the supernatant (~100 ul); add it to the 1.5 mL tube from step 8.
9. Add 0.4 volumes of 7.5M LiCl solution to supernatant from step 8 (final [LiCl] concentration is 2M). Mix well by inverting tubes and precipitate overnight at 4C.
Volumes to use
Day 2
10. Centrifuge solution at 9000g x 20 min, 4C. Discard the supernatant carefully. Let the pellet briefly air dry (5 min) by inverting the tube on towel paper.
11. Thoroughly resuspend the RNA pellet in 400 l of Zymo RNA Lysis buffer, then resume the Zymo process at step 5.
12. Add 320 l of 95 – 100% ethanol (0.8 volumes) to the sample and mix well.
13. Transfer the mixture to a Zymo-Spin IIC column in a collection tube. Centrifuge at > 12000g x 30 seconds. Discard flow through.
14. Add 400 l of RNA Prep Buffer to the column. Centrifuge at > 12000g x 60 seconds. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube.
15. Add 800 l RNA Wash Buffer to the column. Centrifuge at > 12000g x 30 seconds s. Discard flow through, and replace Zymo-Spin IIC column back into the same collection tube. Repeat the wash with 400 l RNA Wash Buffer.
16. Centrifuge the Zymo-Spin IIC column at > 12000g x 2 minutes in the empty collection tube to ensure complete removal of the wash buffer.
17. Carefully remove the Zymo-Spin IIC column from the collection tube and place into an RNase free tube. Add 25 l of RNA Storage Solution directly to the column; let stand one minute.
18. Centrifuge the Zymo-Spin IIC column at 10000g x 30 seconds to elute RNA.
19. Estimate RNA concentration using Qubit.
20. If DNA needs to be removed, treat with DNA-free Turbo kit.
