From 2006.igem.org

Contents 1 Making Cells Competent 1.1 Media/Reagents 1.2 Pre 1.3 Competent Cells: Day 0 1.4 Competent Cells: Day 1 (optional) 1.5 Competent Cells: Day 2

Making Cells Competent

Time: O’N then 3 hr next day

Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl2"

Makes 64x50μL aliquots

Media/Reagents

• LB
• Cells (from plate)
• CaCl2 Solution
• Dry Ice (2nd floor Althouse or S. Frear)

Pre

• 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

1. Streak cells on LB plate
2. Grow overnight at 37°C

Competent Cells: Day 1 (optional)

1. Inoculate overnight starter culture (2 mL) w/ colony from plate

Competent Cells: Day 2

1. 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).
2. Ice for 10 min.
3. Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
4. Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
5. Spin 1100g/5 min/0°C.
6. Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
7. 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.
8. Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
9. Transfer to box(es) and store in -80°C freezer (downstairs on 2nd floor).

¹CaCl2(per L)

• 20 g bactotriptone
• 5 g bacto-yeast extract
• 0.5 g NaCl
• 0.19 g KCl
• Adjust to pH 7.0 w/ NaOH
• Autoclave
• 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