User:Eric M. Walters/Notebook/Spring 2012/2012/04/20

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Checking Addgene plasmids

  • We received pMevT and pMBI (for iGEM/Matt C's stuff) earlier this week
  • I will test that they grow (today) and produce the expected plasmid map (Mon or Tues)
  • I'll streak for isolation, move the plates to the fridge tomorrow, and start O/Ns either Sunday or Monday.

Growth conditions:

  • 37°C on agar plates over the weekend w/ appropriate antibiotic
    • pMevT:Chlormphenicol
    • pMBI:Tetracycline
      • Tet stocks were bad, see below

Plasmid maps (for Monday/Tuesday):

  • pMBI: Given a rough plasmid map (removed ispA from pMBIS), PstI should cut 2x, yield 5.8, 3.9kb bands.
  • pMevT: EcoRV should yield 2 bands, 7.1 & 1.5kb.

Making tetracycline stocks

  • Old stocks were insoluble, made a couple years ago
  • It's a powder in the back of the bottom of the -20°C
  • Dissolved in 190 proof EtOH to 5 mg/mL 1 mL stocks and put in the -20°C
    • Tet selective concentration is 10 μg/mL, so the stocks are 500x (vs normal 1000x)