User:Anthony Salvagno/Notebook/Research/E. coli Transformation Protocol
Today we are inserting the plasmids into E. coli cells. Here is the protocol:
- Need 10ng/uL plasmid DNA
- from a 527ng/uL stock I wanted 200uL stock of above concentration. That means I needed to add 3.8uL of plasmid DNA to 196uL TE buffer.
- Create 4 tubes
- No DNA
- 2.5uL DNA (from 10ng/uL stock)
- 5uL DNA
- 10uL DNA
- combine with 50uL competant E. coli cells.
- Incubate on ice for 30 min
- Heat shock for 1 min at 42C
- Add 1mL LB
- incubate for 60 min at 37C in shaker
- Spin cells at 4krpm for 1 min
- discard ~850uL supernatant
- Mix cells in remaining 150uL
- plate on LB and amp using glass beads
- Incubate plates at 37C overnight
For general protocol modify steps 1, 2, and 3 per user discretion.