Transformation and Preparation for Expression
Transformation of M33S/M103S into Expression Strain
- Take a sterile eppendorf tube and place it on ice.
- Mix 40μL of BL21(DE3) E.coli with 5μL of the M33S/M103S mini-prep (col. 1) 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.
Preparation for Expression
- 1L LB = 25g LB + 1L distilled H2O (combine in 2800mL flask, autoclave on liquid cycle)
- four of these were prepared
- 25mL LB = 0.625g LB + 25mL distilled H2O (combine in 250mL flask, autoclave on liquid cycle)
- four of these were prepared
|
Project name
Main project page
Previous entry Next entry
