Team:iTesla-SoundBio/TransformationofB.Subtilis168

Transformation of Bacillus subtilis 168

1. Inoculate 4 mL of LB media with B. subtilis 168 cells and grow overnight at 37 C, 225 rpm.
2. Dilute the o/n culture to an OD600 of 0.4-0.6 with SM1 to a final volume of 15 mL.
3. Incubate in a sterile flask at 37 C until the culture reaches an OD600 of 2.0-2.8 (this should be around when the culture departs log phase and take around 2 hours).
4. Add equal volume (15 mL) of SM2 to the culture and grow for another 90 minutes.
5. At this point, continue on to transformations or freeze 500 uL aliquots of competent cells with glycerol added to 10% (~55 uL) at -80 C.
6. Add 5 uL of plasmid DNA to 500 uL of competent cells and incubate for another 30 min with shaking.
7. Add 300 uL of LB and incubate for another 30 min.
8. Plate cells on selective LB agar with the appropriate antibiotic.

 

Media

1X ST Base (per L) (Autoclave)	50X YECA (per 50 mL) (Filter Sterilize)
2 g Ammonium Sulfate	5 g Bacto Yeast Extract
12 g Dipotassium sulfate (K2HPO4)	0.625 g Casamino Acids
6 g Monopotassium sulfate (KH2PO4)	Fill to 50 mL with ddH2O
1 g Sodium citrate dihydrate	May need heating to fully dissolve
Fill to 1 L with ddH2O

 

	SM1 (per 15 mL)	SM2 (per 15 mL)
ST Base	~15 mL	~15 mL
MgSO4 (0.3 M)	37.5 uL	150 uL
50X YECA	300 uL	150 uL
Tryptophan (10 mg/mL)	150 uL	150 uL
50% Glucose	150 uL	150 uL
CaCl2 (0.3 M)	-	75 uL

Info for citation:

 

Bennallack et al., J Bacteriol. 2014 Oct 13. pii: JB.02243-14.

PMID: 25313391