Team:Washington/Protocols/Cell Lysate Assay

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=Whole Cell Lysate Assay=
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<!---------------------------------------PAGE CONTENT GOES BELOW THIS---------------------------------------->
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Spin down cells from expression cultures at 4000rpm for 20min, pour off supernatant and perform either Triton Lysis or Sonication Lysis.
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*'''Triton Lysis'''
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**Triton Lysis Buffer (10mL, enough for 2 plates)
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***9mL of 1x PBS
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***10mg of Lsyozyme (5-20 is fine)
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***5mg of DNase (2-10 is fine)
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***1mL of 10% Triton X100
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**Add 50uL of Triton lysis buffer to each well
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**Shake on High Speed Plate Shaker (Set to 1500rpm, in J562) for 20-60 minutes
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**Add 250uL of 100mM NaOAc pH 4.0 to each well
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**Spin 40 minutes 4000rpm
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*'''Sonication Lysis'''
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**Add 300uL of 100mM NHAc pH 4.0 to each well
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***No lysozyme or DNase needed as sonication will break up the cell wall AND the genomic DNA
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**Use Plate Sonicator (Ask Chris)
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**Spin 40 minutes 4000rpm
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*'''2.Assay'''
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**Add 90uL of 5microM substrate (in NaAc pH 4.0 buffer) to each well of a black fluorescent microtiter assay plate
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**Start reaction by adding 10uL Supernatent (try to avoid bubbles and pippette quickly, but accurately)
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***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet!
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**Monitor the reaction with the SpectraMax
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<!---------------------------------------PAGE CONTENT GOES ABOVE THIS---------------------------------------->
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<div style="text-align:center">
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'''&larr; [[Team:Washington/Protocols|Back to Protocols]]'''
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&nbsp; &nbsp; &nbsp;
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</div>

Latest revision as of 01:04, 23 September 2011


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



← Back to Protocols      

Retrieved from "http://2011.igem.org/Team:Washington/Protocols/Cell_Lysate_Assay"