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

• 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