Making Cells Competent
Time: O’N then 3 hr next day
Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl<sub>2</sub>"
Makes 64x50μL aliquots
- Cells (from plate)
- CaCl2 Solution
- Dry Ice (2nd floor Althouse or S. Frear)
- Prepare CaCl2 solution1
- Autoclave centrifuge bottles/tubes and chill on ice
- Before centrifuge steps, make sure centrifuge is on and at 0°C
Competent Cells: Day 0
- Streak cells on LB plate
- Grow overnight at 37°C
Competent Cells: Day 1 (optional)
- Inoculate overnight starter culture (2 mL) w/ colony from plate
Competent Cells: Day 2
- Inoculate 80 mL2 with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency). Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
- Ice for 10 min.
- Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
- Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
- Spin 1100g/5 min/0°C.
- Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
- Spin 1100g/5 min/0°C. Decant supernatant & resuspend well in 1.6 mL ice-cold CaCl2. Cells may stand on ice for 12-24 hrs to increase competency.
- Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
- Transfer to box(es) and store in -80°C freezer.
- 20 g bactotryptone
- 5 g bacto-yeast extract
- 0.5 g NaCl
- 0.19 g KCl
- Adjust to pH 7.0 w/ NaOH
- Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)
2Procedure can be scaled up or down as necessary