Team:University of Ottawa/7 August 2008

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Today in the Lab

Chris

Digestion and Ligation of OA, OB and T Amp Product

  • Digested all three according to previous digestion protocol (see August 5 post). Ran 7 samples--six ligations and one digestion products only. Incubated at 37°C for 15 minutes, followed by 20 minutes at 80°C to denature.
  • Spiked each tube with ligase and ATP then incubated at room temperature for around two hours.
  • Ran products on a 0.6% gel for 1.5 hours at 80V. Stained the gel in an Ethidium Bromide and TAE Buffer solution for 20 minutes.
  • Excised desired band and performed gel extraction. Stored at -20°C.
  • Matt

  • Absorbance was measured from the ten colonies miniprepped by Chris. I then digested each colony with PST1 to confirm.
  • All colonies were confirmed and successful.
  • Used SLN1 primers to amplify the PTP2/GAL10 casette overnight from two colonies chosen from the miniprep.
  • Dan

    p1T miniprep confirmation

  • p1T was digested with HindIII for confirmation, I did not get the correct product :(
  • dehydrogenase construct

  • pSSB37 and pDR197(pure1) were both digested with SphI and HindIII
  • Master plates of pSSB37 and pDR197(pure1) were streaked