User:Dileep D. Monie/Notebook/2008-3-25

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  • Removed plates streaked with 5 test strains and TOP10 from incubator (colonies grew on all plates)
  • Prepared 250 ml of Supplemented M9 Media (this is the same as the Endy Lab version but does not require preparation of intermediate solutions)
    • Mix the following components and autoclave at 121°C for 20 min
    1. 241 ml ddH2O
    2. 5.64 g M9 minimal salts, 5X (Sigma M6030; lot no. 127K1574)
      • ERROR: This amount yields 2X final concentration --DDM, 27 March 2007
    3. 0.5 g casamino acids (Acros 61204-1000; lot no. B0123863)
    4. 1 ml glycerol (Invitrogen 15514-011; lot no. 1248326)
    5. 0.5 ml 1M MgSO4 (Sigma M3409; lot no. 116K6095)
    6. 25 μl 1M CaCl2 (Teknova C0477; lot no. C047710L301Z)
    • Add 7.5 ml of sterile 10 mg/ml thiamine hydrochloride solution
    1. Dissolve 100 mg thiamine hydrochloride (Sigma T1270; lot no. 037K00071) in 10 ml ddH2O
    2. Filter sterilize with a 0.2 μm PES sterile syringe filter
    • Protect Supplemented M9 Media from light (thiamine hydrochloride is light sensitive)
  • Added 5ml of Supplemented M9 Media containing 20 μg/ml Kanamycin (Sigma K0254; lot no. 027K2378) to fifteen 17mm test tubes
  • Added 5ml of Supplemented M9 Media with no antibiotic (for the TOP10 negative control cells) to three 17mm test tubes
  • Inoculated media by picking single colonies from plates (each strain was inoculated in triplicate from different colonies)
  • Grew the cultures at 37°C with shaking at 70 rpm
  • Stored plates streaked with 5 test strains and TOP10 at 4°C