Team:Washington/Protocols/Cell Lysate Assay

From 2011.igem.org

Whole Cell Lysate Assay

Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.

• Triton Lysis
  • Triton Lysis Buffer (10mL, enough for 2 plates)
    • 9mL of 1x PBS
    • 10mg of Lsyozyme (5-20 is fine)
    • 5mg of DNase (2-10 is fine)
    • 1mL of 10% Triton X100
  • Add 50uL of Triton lysis buffer to each well
  • Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes
  • Add 250uL of 100mM NaOAc pH 4.0 to each well
  • Spin 40 minutes 4000rpm

• Sonication Lysis
  • Add 300uL of 100mM NHAc pH 4.0 to each well
    • No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA
  • Use Plate Sonicator (Ask Chris)
  • Spin 40 minutes 4000rpm

• 2.Assay
  • Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
  • Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)
    • Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
  • Monitor the reaction with the SpectraMax
    • Basic Settings = ???