Transforming competent E. coli cells with plasmid
- Don’t do before 3pm; Growth for too long will cause satellite cells to form on plate.
- Obtain an aliquot of chemically competent DH5α cells from the –80 and thaw on ice.
- Once thawed mix 50 uL competent cells with 2 uL plasmid (1–10 ng/uL) and incubate on ice for 20–30 min.
- Heat shock at 42 C for 30 seconds, then quickly place on ice for 5 min.
- Add 250 uL SOC medium and gently mix.
- Put on shaker at 37oC for 1 hour.
- Remove from shaker and add 50–100 uL to each plate – try different volumes (50 vs. 100 uL) to ensure at least one plate will be a good density.
- To make a spreader, take a glass Pasteur pipette and expose the tip to a flame until it bends.
- Flame the spreader by dipping in ethanol and expose to flame. Let cool for 10 seconds before using (if it is too hot it will kill the cells!).
- Pipette cells onto plate and rotate the plate while spreading the cells to distribute evenly .
- Place in 37 C overnight and grab next morning to pick colonies.