User:Anthony Salvagno/Notebook/Research/E. coli Transformation Protocol

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Today we are inserting the plasmids into E. coli cells. Here is the protocol:

Prep

  1. 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.
  2. Create 4 tubes
    1. No DNA
    2. 2.5uL DNA (from 10ng/uL stock)
    3. 5uL DNA
    4. 10uL DNA
  3. combine with 50uL competant E. coli cells.
  4. Incubate on ice for 30 min
  5. Heat shock for 1 min at 42C
  6. Add 1mL LB
  7. incubate for 60 min at 37C in shaker
  8. Spin cells at 4krpm for 1 min
  9. discard ~850uL supernatant
  10. Mix cells in remaining 150uL
  11. plate on LB and amp using glass beads
  12. Incubate plates at 37C overnight

For general protocol modify steps 1, 2, and 3 per user discretion.

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