Transforming Bacillus subtilis
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Streak strain and incubate O/N, and/or inoculate 2mL LB (with antibiotics if necessary)
with a single colony and incubate O/N at RT.
Next morning, inoculate 10mL LB (less for v. small colonies) with antibiotics with either
a single colony or with 100mL O/N culture (OD 600= ~0.2)
Grow to OD 600 ~1 @ 37C (takes about 3 hours). In the meantime, make MD media.
*Remove 1mL MD to use as a blank.
Inoculate 25mL MD with 1.25 mL of the above culture (or 20mL with 1mL culture,
etc.). Do NOT add antibiotics from here on.
Grow for about 4 hours at 37C to reach stationary phase. Cells should now be
competent. Wild type cells attain maximum competence roughly 1­3 hours after
entering stationary phase. For strains with unknown growth curve, plot a growth curve.
Add 50% glycerol to the competent cells to get a final concentration of 20% glycerol.
Divide into 1mL aliquots (easiest to pull up 10mL with a pipet and distribute into eppis).
Freeze in ­80C. It is not necessary to thaw cells on ice.
Transformation: Add DNA (2­5mL chromosome, 5­10mL plasmid) to 200mL
competent cells and incubate on the rotary shaker or roller drum for 30m @37C.
Plate appropriate dilutions on plates containing the appropriate antibiotics.
To make MD medium (check your strain’s auxotrophy!):
In a flask, combine:
10mL MD
20mL MD
30mL MD
50mL MD
1X PC buffer
9.12 mL
18.24 mL
27.36 mL
45.6 mL
Glucose (50% w/v)
400 mL
800 mL
1200 mL
2.0 mL
L­tryptophan (10mg/mL)
100 mL
200 mL
300 mL
500 mL
Ferric ammonium citrate
50 mL
100 mL
150 mL
250 mL
(2.2 mg/mL)
Potassium aspartate*
250 mL
500 mL
750 mL
1.25 mL
(100 mg/mL)
1M MgSO4
30 mL
60 mL
90 mL
150 mL
Phenylalanine (10 mg/mL) 50 mL
100 mL
150 mL
250 mL
*Can also use potassium glutamate but is slightly less efficient
Tip: When diluting 10X PC buffer for i.e. 10mL MD, make 10mL 1X PC, and remove the extra
0.88mL.
1L 10X PC (Phosphate­citrate) buffer:
107 g
60 g
10 g
to 1L
K2HPO4 (dibasic anhydrous)
KH2PO4 (monobasic anhydrous)
Sodium citrate • 2H2O
dH2O
Dilute stock solution, check pH of 1X PC buffer and adjust (if necessary) to pH 7.5.
(1X PC approximately corresponds to Spizizen’s salts without ammonium sulfate)
Autoclave