Making Competent Cells

Transformation Buffer 1 (TFB1): all concentrations final

30 mM potassium acetate
50 mM manganese chloride
100 mM potassium chloride
10 mM calcium chloride
15% v/v glycerol

pH to 5.8 with diluted (0.2 M) acetic acid

**throw away if overshoot pH 5.8
filter sterilize & store @ 4˚C

Transformation Buffer 2 (TFB2): all concentrations final

10 mM sodium MOPS pH 7.0

75 mM calcium chloride
10 mM potassium chloride
15% v/v glycerol

pH to 7.0 with diluted (0.2 M) NaOH

**throw away if overshoot pH 7.0
filter sterilize & store @ 4˚C
 1. Start overnight LB culture (2-5 mL)
-make sure have 2-50mL LB flasks ready for
tomorrow
-sterile labeled 1.5 mL tubes in racks @ -80˚C
2. Add overnight culture to each LB flask
3. Shake @ 37˚C until O.D. @ 600 nm = 0.5-0.6
4. Transfer culture to pre-cooled 50 mL centrifuge tubes
5. Spin cells down in pre-cooled 50 mL centrifuge tubes
at 2500 rpm/4˚C for 15 minutes
6. Remove supernatant
7. Add 12.5 mL TF1 Buffer to each tube
-GENTLY resuspend the pellet using the pipet
-incubate cells on ice for 30 minutes
8. Spin cells down at 2500 rpm/4˚C for 15 minutes
9. Remove the supernatant
10. VERY VERY GENTLY add 4 mL TF2 Buffer to each
pellet. VERY VERY GENTLY resuspend cells.
(always keep butt of tube close to ice)
11. Now aliquot cells in -80˚C pre-cooled 1.5 mL tubes
12. Store tubes @ -80˚C