Isolation of RNA from tuber by LiCl-SDS-Phenol procedure

• Day-1

  • Grind 1.5 g of tissue in liquid nitrogen. Immediately transfer the material to a 50 ml centrifuge tube containing 15 ml grinding buffer + 5 ml TLE equilibrated phenol.
  • Homogenize the mix with polytron at moderate speed.
  • Add 5ml chloroform.
  • Repeat polytron homogenization at low speed.
  • Incubate at 50 c for 20 min.
  • Centrifuge at 10,000 rpm for 10 min at 4 C.
  • Pipette out aqueous phase, add 5 ml equilibrated phenol. Mix by inversion, add 5 ml chloroform, mix by vigorous shaking.
  • Centrifuge at 10000 rpm for 15 min at 4 C.
  • Take out aqueous phase and repeat phenol-chloroform extraction twice more (usually three extractions).
  • Extract aqueous phase with equal volume of chloroform.
  • Transfer aqueous phase to a clean 50ml tube, add 8M LiCl (1/3 vol) to bring solution to a final concentration of 2M LiCl.
  • Incubate at 4 C overnight for RNA precipitation.
• Day-2
  • Centrifuge at 10000rpm for 20 min at 4 C.
  • Carefully rinse pellet with a 1-2 ml of 2 M LiCl (without disturbing the pellet).
  • Resuspend pellet in 500 m l water.
  • Add 8M LiCl to bring conc. of LiCl to 2M, incubate at 4 C (ice bath) for atleast 2h for RNA precipitation.
  • Centrifuge at 10000 rpm for 20 min at 4 C.
  • Rinse pellet with 200 m l of 2M LiCl. Resuspend pellet in 200 m l of water.
  • Add 20 m l of 3M sodium acetate, pH 5.2 and 2.5 vol of absolute ethanol. Incubate at -20 C overnight.
• Day-3
  • Centrifuge at 10000rpm for 15 min at 4 C. Wash pellet with 75% ethanol.
  • Air dry the pellet and dissolve in 20-30 m l of water (During Northern one vol of RNA and 3 vol of loading dye has to be added, so it is desirable to have high conc. of RNA).
  • Check RNA on 2% gel.

RNA extraction solution

• Grinding buffer (LiCl, pH 8.0) 500 ml

  • Tris 0.18M 10.85 g

  • LiCl 0.09M 1.905 g

  • EDTA 4.5 mM 0.835 g (can be added from stock solution)

  • SDS 5.0 g

• TLE solution 2Lt

  • 0.2M Tris 48.44 g

  • 0.1M LiCl 8.44 g

  • 0.5M EDTA 3.72 g

  Adjust pH to 8.2 with HCl. This is used to equilibrate phenol.

  • 8M LiCl – 50 ml: 16.9 g
  • 2M LiCl – 50 ml: 2.11 g

Solution be prepared with RNase-free water. Add 1 ml of DEPC to 1 lt of dd water, shake vigorously and autoclave for 40-60 min. Keep this water in a sterile environment for 1-2 day for DEPC to get inactivated.

Solution 2-4 can be autoclaved, but there is no need to autoclave soln. 1.