IGEM:UC Berkeley/2006/Plasmid Transformation

KCM-competent cell heat shock

1. create cocktail on ice

  *20 uL TGI cells (for a cotransformation use 100uL of cells)

  *10 uL KCM buffer

  *70 uL water

  *1 uL plasmid (for cotransformation use 2uL of each plasmid)

2. heat shock for 1.5 min at 42 C 

3. incubate on ice for 5 min. 

3.5 If antibiotic marker is anything but Amp then rescue cells in ~100uL (or about equal volume) LB for one hour at 37C.

4. plate 

-JL_06/09/06

Transformation of Ligation mix

1. Create cocktail on ice

  *100 uL TGI cells

  *15 uL KCM buffer

  *25 uL water

  *10 uL plasmid

2. Incubate on ice for 10 min.

3. Heat shock for 1.5 min at 42 C 

4. Rescue: add 150uL of SOB media 

5. Incubate in 37 C shaker for 1 hour

6. Plate

-JL_06/14/06

Electro Shock Competence

1. Set volts: 1.60 (slow thaw cells on ice) Use 2IT media or SOB media

2. Put 50 mL of cells into clean eppendorf and limit bubbles. Add 1 uL of ligation mixture and stir to mix.

3. Pipette into electro cuvette with its cap off. Tap down to bottom, dry the tube

4. Put into holds, well seated in back 

5. After beep, rescue with 500 mL(have ready) of media. Transfer all volume back to eppendorf, no longer on ice.

-SIL_06/19/06