STEWART LABORATORY

 

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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 4C

 

                              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 -80C