Griffitts:Trizol purification

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Procedure

  • Grow up Rm1021 (B100) to OD600 = 0.9–1
  • Place the cells on ice to inhibit further growth
  • Centrifuge 1 mL of cells for 1 minute
  • Dump and tap
  • Add another 1 mL of cells and centrifuge for 1 minute
  • Remove supernatant and store pellet on ice DUMP AND TAP?
  • Make TED solution
  • Add 50 μL of TED solution to the pellet while on ice
  • Mix well
  • Add 50 μL of 1% SDS to the pellet
  • Immediately vortex 1 second
  • Place in 68°C heat block for 3 minutes
  • Add 1 mL trizol (in fume hood)
  • Vortex until pellet is broken apart
    • About 1 minute
  • Add 200 μL chloroform (in fume hood) to tube
  • Shake vigorously
  • Let stand for 5 minutes
  • Centrifuge at full speed for 2 minutes
  • Carefully remove 600 μL of the upper phase to a new tube
  • Add 600 μL of isopropanol (in fume hood)
  • Invert to mix
  • Place on ice for 10 minutes
  • Centrifuge for 5 minutes
  • Dump and tap
    • There should be a white precipitate at the bottom
  • Wash with 300 μL of 75% ethanol
  • Invert to mix
  • Centrifuge
  • Carefully remove ethanol
  • Store pellet in the -20°C or -80°C freezer

When ready, proceed to DNAse treatment

Solutions

TED

Keep on ice

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