Hot Phenol RNA isolation

RNA isolation from Salmonella typhimurium or other gram-negative bacteria.

  1. Bacterial culture is grown to wanted OD600.
  2. T0 sample is removed and processed as below, remainder is transferred to specialised conditions and incubation is continued and then processed as below.
  3. Pipet 100ml of bacterial culture into a 250ml centrifuge bottle containing 16ml Stop Solution.
  4. Spin for 2 minutes at 8000rpm in Beckman centrifuge at 4°C.
  5. The supernatant is carefully and completely removed from tubes by decanting and gentle tapping to remove any remaining liquid.
  6. Freeze pellet by addition of just enough liquid nitrogen to cover pellet when tube is tilted, use only screw cap without seal and put it on loosely, store at -80°C until further purification.
  7. Resuspend the cells by adding 10ml fresh lysozyme solution and vortexing.
  8. Transfer to 40ml plastic reusable centrifuge tubes rinsed with chloroform and dried.
  9. Add 1ml 10% SDS and mix by vortexing.
  10. Incubate in 64°C water bath for 1-2 minutes, until sample clears.
  11. Add 11ml 1M sodium-acetate using flamed glass pipet and mix by vortexing.
  12. Add 22ml of phenol using flamed glass pipet, invert 10 times.
  13. Incubate at 64°C for 6 minutes with 6-10 inversions every 40 seconds.
  14. Chill tube on ice.
  15. Centrifuge for 10 minutes at 10,000rpm (4°C).
  16. Transfer aqueous layer using 1ml pipetman to a new reusable centrifuge tube rinsed with chloroform containing 22ml (equal volume) chloroform.
  17. Invert 6-10 times.
  18. Centrifuge for 5 minutes at 10,000rpm (4°C).
  19. Transfer aqueous layer to a new reusable centrifuge tube rinsed with chloroform and dried.
  20. Add 2.2ml (1/10th volume) 3M sodium-acetate, EDTA.
  21. Add 22ml (equal volume) isopropanol.
  22. Place in -80°C freezer for 20 minutes or overnight.
  23. Centrifuge at 14,000rpm for 25 minutes at 4°C.
  24. Remove supernatant carefully, leaving a white pellet.
  25. Add 40ml 80% Ethanol to pellet (fill tube).
  26. Centrifuge at 14,000rpm for 25 minutes at 4°C.
  27. Remove supernatant carefully and air-dry pellet for 15-20minutes in fume hood.
  28. Resuspend pellet in 2ml water (DEPC treated).
  29. Split sample into two 2 ml centrifuge tubes.
  30. Add 12.5µl of RNase inhibitor (40 U/µl).
  31. Add 25µl of DNase 10U/µl.
  32. Add 20µl of 1M Tris (pH 8.3).
  33. Add 10µl of 1M MgCl2.
  34. Mix and incubate at 37°C for 30 minutes.
  35. Add 1ml phenol and invert 6-10 times.
  36. Centrifuge for 2-3minutes at full speed in micro-centrifuge.
  37. Transfer clear upper layer to new tube containing 1ml (equal volume) of phenol-chloroform.
  38. Invert 6-10 times.
  39. Centrifuge for 2-3minutes at full speed in micro-centrifuge.
  40. Transfer upper layer to new tube.
  41. Add 1ml (equal volume) chloroform and invert 6-10 times.
  42. Centrifuge for 2-3minutes at full speed in micro-centrifuge.
  43. Transfer upper layer to new tube.
  44. Add 1ml (equal volume) chloroform and invert 6-10 times.
  45. Centrifuge for 2-3minutes at full speed in micro-centrifuge.
  46. Transfer upper layer to new tube and aliquot as 100µl volumes in new tubes.
  47. Add 10µl (1/10th volume) of 3M Sodium acetate.
  48. Add 200µl (2 volumes) of cold isopropanol.
  49. Incubate at -80 °C for 20minutes or overnight if required.
  50. Centrifuge at 14,000rpm for 25minutes in micro-centrifuge in cold room.
  51. Wash pellets by adding 0.3ml 80% Ethanol.
  52. Centrifuge briefly at 14,000rpm.
  53. Remove ethanol and dry pellets at room temperature.

    The RNA can be stored in -80°C freezer. Resuspend in 30µl DEPC-treated water.

    Control of RNA

Solutions

Phenol, RNA-grade
RNA-grade phenol from Sigma (Catalog number P4682), or other RNase free water-saturated phenol solution with a pH of under 7.
Stop Solution
5% phenol in 95% Ethanol.
Fresh Lysozyme solution
0.5mg/ml lysozyme, TE pH 8.0.
1M Sodium-acetate
300 ml 3M NaOAc, pH 5.2, 600ml dH2O, and 0.9ml DEPC - incubate at 37° overnight and autoclave.
3M Sodium-acetate, 1 mM EDTA
0.5l 3M NaOAc (pH 5.2), 1µl 0.5M EDTA, and 0.5ml DEPC - incubate at 37° overnight and autoclave.
80% Ethanol
420ml 95% Ethanol, 80ml DEPC-treated water.
Water, DEPC-treated
1l of dH2O, and lml DEPC - incubate at 37° overnight and autoclave.
1M Tris (pH 8.3)
0.4l DEPC treated water, 60.5g Tris (from separate container for RNA work), calibrate pH-meter, rinse probe in 0.1M NaOH, 1mM EDTA, followed by DEPC-treated water, measure pH and adjust to pH 8.3 with concentrated HCl, make up to 0.5l with DEPC-treated water.
Phenol-chloroform
50% phenol, and 50% chloroform mixed in centrifuge tube.
3M sodium acetate
0.5l 3M NaOAc pH 5.2 (DEPC-treated), and 0.5ml DEPC - incubate at 37° overnight and autoclave.

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