Team:Paris Liliane Bettencourt/Notebook/2011/07/27/

From 2011.igem.org

Team IGEM Paris 2011

Follow growth of B. subtilis in MDCH medium

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MDCH medium

  • Phosphate-citrate buffer (10 x PC) 10 x concentrated stock solution contains per liter : K2H PO4 (anhydrous) 107 g; KH2 PO4 (anhydrous) 60 g; Trisodium citrate (5 H2O) 10 g.
  • Dilute stock solution, check pH of 1 x PC buffer and adjust (if necessary) to pH=7. (1 x PC corresponds to Spizizen's salts without ammonium sulfate)
  • 10 ml MD medium : 1 x PC buffer 9.2 ml; Glucose (50 %, w/v) 0.4 ml; L-tryptophan (5 mg/ml) 0.1 ml; Ferric ammonium citrate (2.2 mg/ml) 0.05 ml; Potassium aspartate or potassium glutamate (100 mg/ml) 0.25 ml; 1 M MgSO4 0.03 ml.
  • Add 0,2mL of casein hydrolysate in the MD medium
  • A 10 ml preculture is grown overnight at room temperature in LM broth (liquid LB medium supplemented with auxotrophic requirements and 3 mM MgSO4). MDCH medium is prepared by adding 0.2 ml 5 % casein hydrolysate to 10 ml MD. Use the preculture to inoculate MDCH medium at an OD600 of about 0.05. Grow this culture at 37° C with shaking to To (transition from exponential to stationary phase). Sometimes, To do not appears and we can see a decrease of the OD: do not use the culture. Advice: launch 2 or 3 culture for each strain.
  • Add 1 volume of fresh MD medium without casein hydrolysate to 1 volume of culture. Continue shaking at 37° C for 1 hour.
  • Make aliquots of 500μL
  • Add DNA (up to 1μg) and continue shaking in the presence of DNA for 20 minutes. Eventually, allow for expression of antibiotic resistance for 20mn (as described inJ. Bacteriol. (1988), 170 : 5093-5101) and plate.
  • Cm: 5μg/mL; Spec: 50 to 100μg/mL; Kan: 5μg/mL, Erm: 1μg/mL with 25μg/mL of Lincomycin; Tet: ?μg/mL
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