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Team:Washington/Protocols/Cell Lysate Assay
From 2011.igem.org
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***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet! | ***Use the P20 multichannel with a taped cardboard stopper to make sure you don't hit the pellet! | ||
**Monitor the reaction with the SpectraMax | **Monitor the reaction with the SpectraMax | ||
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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
- Triton Lysis Buffer (10mL, enough for 2 plates)
- 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
- Add 300uL of 100mM NHAc pH 4.0 to each well
- 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
