STEWART LABORATORY

COMPETENT CELLS FOR BACTERIAL TRANSFORMATION

REAGENTS

RF1 RF2

COMPOUND	AMOUNT/LITER	FINAL CONCENTRATION (PER LITER)
RbCl	12 g	100 mM
MnCl2 · 4H2O	9.9 g	50 mM
K Acetate	30 mL of a 1M stock (pH 7.5)	30 mM
CaCl2 (dihydrate)	1.47 g	10 mM
Glycerol	150 g	15% (w/v)
Adjust the pH to 5.8 with 0.2 M acetic acid. Sterilize by filtration through a pre-rinsed 0.22 μm filter

COMPOUND	AMOUNT/LITER	FINAL CONCENTRATION (PER LITER)
MOPS	20 mL of 0.5M Stock (pH 6.8)	10 mM
RbCl	1.2 g	10 mM
CaCl2 (dihydrate)	11 g	75 mM
Glycerol	150 g	75 mM
Adjust the final pH to 6.8 with NaOH (if necessary). Sterilize by filtration through a pre-rinsed 0.22 μm filter

PROCEDURE

All manipulations should be done on ice and in the cold room

1. Pick a single colony into 2 mL LB broth for an overnight culture, no antibiotics

2. Dilute 0.5 mL into 100 mL LB broth and culture (~3 hr). Save some of the same broth for a negative control

3. Read OD600. Should be between 0.4 and 0.6

4. Incubate on ice for 15 min

5. Centrifuge at 3000 rpm for 15 min in 50 mL conicals at 4°C

6. Remove supernatant, re-suspend in 33 mL RF1

7. Incubate on ice for 15 min and then centrifuge as before

8. Remove supernatant, re-suspend in 8 mL RF2

9. Aliquot 100 μL per 1.5 mL eppendorf tube

10. Snap freeze into liquid nitrogen and store at -80°C