User:David Johnston Monje/Notebook/Maize Endophyte Biofertilizers/2010/07/16

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The Daily Grind

  • To do: Make 150 mL R2A plates of 0, 500, 1000, 2,000, 5,000, and 10,000 ug/mL arabinose - leave til Monday
  • To do: Array Arabad::GFP in pDRIVE and pDSK-GFPuv tagged JM109, 1A1, 1A8, 1B12, 1C4, 1C8, 1E9, 1E10, 1E12, 1F7, 1G6, and Kp342 into 96 well PCR plate (colony resuspention) and imprint on the above plates. Did this with OD=0.1 bacteria.
  • To do: Dip potatoes in the above microbes (both types of plasmids). They were all at OD=0.1.
  • To do: Resuspend pDSK-GFPuv tagged JM109, Kp342, 1B8, 1C4 (LARGE COLONIES – E.coli), 1C12, 1E3, 1E9, and 2C10 to OD600=0.1 and inject 25 ul into two plants downstairs at 5 cm above crown. Also do two sterile buffer injections.
  • To do: Book microscope for Wednesday (5 days post innoculation)
  • To do: Book car for July 26 trip to London
  • Only injected 5 leaf stage plants (at 10 pm on Friday), so only did one rep of pDSK-GFPuv tagged endophytes today. Injeted 10 ul into each stem twice (instead of 25 ul once which was too much for stems to hold). Noticed lots of exudate coming out of plants, and now realize unless I set up something really specific and contorlled for rhizosphere sampling, there's contamination and I can't pretend that the GFP tagged endophytes aren't there because of dripping. So I will only focus on localization of endophytes and not on proving they are exiting the plant. May have to sample just above the crown (microscopically) as well as roots to prove they are travelling through center of the stem and not just dripping down to the roots.



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