Griffitts:Trizol purification

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

• 880 μL ddH₂O
• 40 μL 0.5 M Tris pH 7.5
• 40 μL 0.25 M EDTA ph 8
• 40 μL 1 M DTT (from -20°C stock)

Keep on ice