Team:UNIPV-Pavia/Notebook/Week17

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Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14
Week 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21



Week 17: 09/8/08 - 09/12/08

09/8/08

  • We prepared 0.5 l of LB + Amp for liquid cultures.
  • We infected 9 ml of LB + Amp with 30 µl of Lig.30(3), Lig.31, Lig.22, Lig.13 and two falcon tubes for Lig.a.
  • We received 3OC6HSL from Sigma! we resuspended it in ddH2O, we prepared some stocks and stored them at -20°C.

09/9/08

  • Glycerol stocks/miniprep for Lig.30(3), Lig.31, Lig.22 and Lig.13.
  • Plasmid digestion for:
    • Lig.30 (S-P)
    • Lig.31 (S-P)
    • Lig.30 (X-P)
    • Lig.22 (X-P)
    • 13 (X-P)
  • Run/gel extraction.
  • Ligations:
    • Lig.31-Lig.30 (="Lig.34")
    • Lig.31-Lig.22 (="Lig.35")
    • Lig.30-Lig.13 (="Lig.T5" for green fluorescence test)
  • We induced one of the two Lig.a overnight culture with 3OC6HSL 1 µM. We incubated the two cultures for 1 hour and then watched TRITC channel at microscope. (We didn't synchronize the two cultures, but performed a qualitative test for luxR mutated protein integrity evaluation).
  • Fluorescence test results: HSL induced Lig.a show RFP expression, while Lig.a without HSL showed a weak red fluorescence. Result pictures are not available at the moment.

09/10/08

  • We transformed/plated ligations. We decided to perform two transformations for each ligation:
    • one normal transformation (1 µl of ligation);
    • one diluted ligation (1:10)
  • We decided to try diluted transformations to have less colonies in the plate and to avoid streaking single colonies plates when colonies are not insulated.

09/11/08

  • Plates grew correctly and diluted transformation showed less colonies.
  • Colony PCR for Lig.34, Lig.35 and Lig.T5 (4 colonies for normal transformation plates and 4 colonies for diluted transformation plates).
  • Gel results (gel picture not available):
    • Lig.34
    • Lig.35
    • Lig.T5