Transformation of Triple Mutant into Expression Strain and Sonication of Double Mutant Expressed Cells
Transformation of M33S/M103S/M8S into Expression Strain
- Take a sterile eppendorf tube and place it on ice.
- Mix 30μL of BL21(DE3) E.coli with 5μL of the M33S/M103S/M8S mini-prep (col. 3-sequence confirmed) in the cold, sterile tube.
- Incubate this mixture for 30 minutes on ice.
- Transfer the tube to a heat block at 42°C for 90 seconds, and then transfer the tube back to ice for 5 minutes.
- Add 200μL of SOC media to the cells/PCR product.
- Shake the mixture at 250rpm at 37°C for one hour.
- Spread 100μL of the mixture on an LB plate with 100μg/mL ampicillin.
- The LB plate was made with 0.875g LB, 0.7g Agar, and 35mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 35μL of 100mg/mL ampicillin was added to it and it was poured into a sterile petri dish.
- Incubate the plate overnight (inverted) at 37°C.
Sonicate M33S/M103S BL21(DE3) E.coli to extract protein
- Safety First: Make sure to wear noise blocking earmuffs.
- 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.
- Distribute sonicated cells between 6 centrifuge tube for a balanced spin.
- Centrifuge the cells for 2 hours at 4°C at 18000rpm.
- Pour off and store supernatent at 4°C.