Transformation Protocol

Protocol

 

 

* Materials

 

- Competent cell (DH5α)

- Ampicillin

- LB plate

  1) Dissolve LB-Agar (40g/L) in DW and autoclave

  2) Once the temperature drops to 45-50℃, add Ampicillin at a final concentration of 100ug/ml

  3) Before the LB-Agar solution solidifies, pour 15~20ml each into the 90-100mm petri dish

  4) Dry the plate upside-down, O/N

  5) Store the plate in 4℃

 

 

* Methods

 

* Make sure the LB plate is warmed enough (at least RT)

* Never Vortex Cells (Just gently tap the tubes)

 

1. Melt Competent Cells on ice

2. Add 200λ of SOC media

3. Add 1λ of Plasmid DNA (10~100ng)

4. Wait for 5 min, on ice

5. Heat shock, 42℃, 45~60s

6. Stabilize for 2 min on ice

7. Seed 100λ of competent cells with a spreader on the LB/Amp+ plate

8. Incubate in 37℃ incubator, O/N