TSS Transformation
Strains: Transformant strain
Reagents: TSS buffer, plasmid, LB plates, antibiotics
Time required: 4-5 hours (excluding Day 0)
A. pcp20 Transformation
Day 0
- Inoculate a 3ml overnight culture of transformant strain in LB supplemented with any antibiotics required for plasmid/insert maintenance.
Day 1
- Dilute the overnight culture 1:100 in 3mL LB supplemented with antibiotics and incubate at 37C for 1-2 hours.
Day 1
- After 1-2 hours, once the culture is translucent but not opaque, swirl the culture in ice water bath for 5-10 minutes to stop cell growth.
Day 1
- Centrifuge the culture in two 1.5mL centrifuge tubes at maximum speed for 5 minutes to get a cell pellet. Resuspend the cell pellet in 100uL TSS buffer.
- One of these tubes can be used as a control and will get no plasmid DNA.
Day 1
- Add 1uL plasmid DNA to the tube and incubate on ice for 30 minutes.
- Set a desk-top heater to 42C for the heat-shock step.
Day 1
- Heat shock the cells twice by doing two 45 second heat exposures with a two minute gap on ice (45 sec shock \(\rightarrow\) 120 second on ice \(\rightarrow\) 45 sec shock). After these heat-shocks, let the tubes incubate on ice for 2 minutes and add 500uL SOC to each. Incubate the cells at 37C (or another temperature required for plasmid stability) for 45 minutes.
- During this time, make selection plates for plating the transformants (with previous and new antibiotics)
Day 1
- Plate 100uL of the culture on selective plates. Grow the plates overnight at 37C (or alternative temperature for plasmid).