Neither plate had growth overnight. We will need to continue to ponder this problem.
Extraction and Sonication of Expressed Cells
- Defrost both sets of cell that were frozen yesterday (wild-type Asc Hb in BL21(DE3) and the triple mutant in the same strain)
- Add 11mL of lysis buffer (25mM HEPES, 5mM imidazole, 500mM NaCl) per liter of LB the cells were grown in (so in this case, 22mL for each protein-type)
- 500mL of this buffer was prepared by adding 2.98g of HEPES to dH2O, the buffer was brought to pH 7.5 with NaOH, 14.61g of NaCl and 0.17g of imidazole were added to the buffer, the final volume was increased to 500mL with dH2O, and the buffer was vacuum filtered
- Safety: Make sure to wear noise blocking earmuffs for sonication.
- Sonicate each tube for 30 seconds at power setting 11.
- Place each tube on ice for at least 30 seconds after sonication.
- Repeat this procedure two more times per tube.
- Distribute sonicated cells between 4 centrifuge tubes for a balanced spin.
- Centrifuge the cells for 2 hours at 4°C at 18000rpm.
- Pour off and store supernatent at 4°C.