Team:Freiburg/Protocols/Cell Lysis
Cell disruption
LP to disrupt E. coli cells by sonification and separate supernatant and pellet
material: PMSF, abs. ethanol, Lysis buffer
duration: ~ 2h with 10 samples
- Prepare 200 µL 500 mM PMSF (phenylmethylsulfonyl fluoride) (17.4 mg PMSF + 200 µL abs. ethanol)
- resuspend frozen pellets in 10 mL Lysis buffer (as good as possible)
- Disrupt cells by 5 min sonication on Bandelin sonoplus with 60 % sonification frequency and 50-70 % power
- add 20 µL PMSF
- centrifuge 30 min @ 10000 g 4°C
- separate supernatant
- centrifuge supernatánt 20 min @ 16000 g 4°C
| buffers | |
|---|---|
| standard buffer/lysis buffer | 20 mM Tris pH 7.5 |
| 150 mM NaCl | |
| 0.3 mM PMSF | |
| 10mM Imidazole | |
