Yale RNA prep
互联网
496
Yale RNA prep
(Rebecca D. Burdine, Michael J. Stern [modified by S.M. Hettenbach])
- Add 8 ml of TRIZOL to 2 ml packed worms in 15 ml centrifuge tube. Vortex and invert tube to solubilize and lyse worms for at least 10 min.
- Divide into two 15 ml c/f tubes--5 ml each.
- Invert for additional 5 min. Solution should be pink and will contain thread-like material. Spin at 4°C, 4000 rpm for 20 min to remove insoluble material. All spins done in fixed angle rotor with white inserts.
- Transfer supernatant to new 15 ml c/f tubes(2). Add 800 l chloroform to each tube.
- Vortex 15 sec, incubate at room temperature for 3 min. Smeary preps will result if vortexing not long enough.
- Spin at 4°C, 4000 rpm for 20 min to separate phases.
- Transfer upper phase to new 15 ml c/f tubes(2). Add 2 ml isopropanol to each tube.
- Invert to mix & incubate at RT for 10 min. Spin at 4°C, 4000 rpm for 30 min.
- Remove s/n with pipet without disturbing the pellet.
- Add 400 ul 75% EtOH, vortex briefly.
- Spin at 4°C, 4000 rpm for 10 min.
- Remove s/n with pipet and invert racked tubes in hood with door half shut. Approx. 10-15 minutes; do not overdry.
- Add 300 l DEPC-treated ddH2 O to each tube and resuspend and combine tubes using wide opening tips. May have to heat briefly at 45-50°C to completely dissolve. Really good yields may require adding more H2 O.
- Use 2 l to do OD260 and OD280 to determine concentration. An A260/280 ratio of <1.6 indicates partially dissolved RNA. 1 OD260 is equal to 40 g/ml of RNA.
- Store at -80°C in 1 mg aliquots, avoid repeat freezing and thawing.
