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Further Adventures in Ligation & some black smelly agar

TSI Agar slants

  • Slant inoculated with the LE392 transformants containing pSB74 showed a mass of black precipitate near the surface, definitively confirming the production of hydrogen sulfide (!), the smell of which was by now very strong. The DH5alpha transformants did not exhibit the same H2S odor.
    blurry (apologies!) image of LE392 slant showing FeS precipitate(right) next to un-inoculated slant (left)

Ligation Attempt #2

  • Still don't know whether ligation attempt #1 was successful, as colony PCR on 7/1 was not definitive. Will sequence the results for a certain answer, but given the small number of colonies think it best to redo ligation in the interim in case of failure.
  • To this effect redid double digestion of phsABC insert with EcoRI and SpeI and the serial digestion of B0015 with EcoRI and then XbaI. Simultaneously digested J23114 to be inserted into P0312, which lacks a promoter. Each digestion reaction had a total volume of 100 uL and included 2 ng of DNA, double the amount used previously, so that even after purification there would still be a sizable amount of DNA remaining for ligation. Otherwise digestion and purification was run the same as on 6/29 & 6/30.

This day's activities are also recorded on pages 42-45 of the hard copy lab notebook