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RNA Extraction from Endosperm

RNA Extraction From Endosperm SDS-Trizol combo
(based on a protocol by Joao Leiva, Ricardo Dante and David Holding)

SDS Extraction (based on Prescott and Martin 1987)

    1. Add 100mg ground endosperm to a tube with 200ul RNA extraction buffer.

        To make 200ml Buffer:

  • 10ml 1M Tris
  • 30ml 1MLiCl
  • 2ml 0.5M EDTA
  • 118ml H20
  • DEPC treat, and autoclave
  • add 40ml 5% SDS

 

    2. Add 200ul of 1:1 Phenol-Chloroform, shake it well and place them on ice for 5 min.           mix occasionally.
    3. Transfer Solution to a Phase Lock tube (Eppendorf) (pre-spin the Phase Lock                    tubes for 30 sec at 12000g before adding solution. Never vortex Phase Lock tubes.
    4. Centrifuge the samples for 10 min @ 10000g, 4 C.
    5. Add 200ul of Phenol-Chloroform, Shake well
    6. Centrifuge samples for 10 min at 10000g, 4 C.
    7. Add 200ul of Chloroform, shake well, and place on ice for 5 min. Mix occasionally.
    8. Centrifuge samples for 10 min at 10000g, 4 C.

TRIZOL Extraction

  1. Pour the aqueous phase into a new Phase Lock tube (the organic phase will be locked in the bottom of the tube)
  2. Add 1 ml Trizol.
  3. Shake well for 15 sec. Incubate for 5 min at room temp.
  4. Add 200ul of Chloroform, shake it well and incubate for 2-3 min at room temp.
  5. Centrifuge for 10 min at 10000g, 4 C.

 

RNA Precipitation

  1. Pipet 700ul of aqueous phase to a fresh 1.5ml microfuge tube.
  2. Add 700ul of Isopropanol, mix well, and incubate for 10 min on ice.
  3. Centrifuge for 10 min at 10000g, 4 C.(Precipitate can be seen at this point)

 

RNA Washing and Dissolving

  1. Pour out the supernatant
  2. Add 1 ml of 70% EtOH (in DEPC treated water)
  3. Centrifuge for 5 min at 10000g, 4 C.
  4. Carefully pour out the supernatant.
  5. Air dry the RNA pellet.
  6. Re suspend in 50ul of DEPC treated water