20.109(F09): TA's notes for module 3

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Friday November 6

  • Make up overnight inoculum of phage for Thursday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight
  • autoclave/sterilize 16 125-mL flasks (one flask per group)
  • make sure there are enough Oak Ridge tubes for each group (=3 per group)
  • make sure that there is a centrifuge available for Thursday and Friday afternoon for the PEG precipitation (Niles lab down the hall?)
  • make enough PEG solution (20% PEG-8000, 2.5 M NaCl --- this requires a long time to fully solubilize - leave on the stir plate for about an hour)


Saturday November 7

  • Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks (for Thursday class). Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Monday night)


Sunday November 8

  • Make up overnight inoculum of phage for Friday class: 10 mL LB, 10 µL XL1-blue, 10 µL tet, 10 µL virus (from supernatant or PEG precipitated); rotate at 37ºC overnight


Monday November 9

  • Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Thursday's class)
  • Aliquot 40 mL LB and 40 µL Tet into each eight 125-mL flasks. Add 1 mL of overnight inoculum, and incubate shaking at room temperature for two days (til Wednesday night)


Tuesday November 10

  • check to make sure there are enough LB plates for each group to have 4 total
  • check to see that there is enough top agar for each group to have 12 mL total


Wednesday November 11

  • Take off 8 125-mL flasks with 40 mL of now-saturated phage culture. Store at 4ºC (this will be for Friday's class)
  • Use small (yellow-capped) tubes to make titering cells - about 1 mL is necessary for each group, so make up 6 5-mL cultures with 5 mL LB, 5 µL Kan, and a colony of ER2267. Incubate rotating overnight at 37ºC


Set up for Day 1 - Thursday November 12th and Friday November 13th

  • ice buckets (three) filled with ice for PEG precipitation
  • 40 mL phage cultures
  • titering cells (ER2267)
  • necessary chemicals (CTAB, hydrated hydrogen tetrachloroaurate, silver nitrate, ascorbic acid, and NaCl) - check their calculations before they proceed
    • Each group should make a 0.1 M solution of hexadecyltrimethylammonium bromide ("CTAB"). This is done by mixing 0.72 g with 20 ml of water in a 50 ml conical tube - CTAB will be placed in 30ºC incubator the day before to fully solubilize
    • One group will be responsible for making each of the following solutions that the class will use in the synthesis of nanowires.
      • 10 mM hydrated hydrogen tetrachloroaurate (HAuCl4•3H2O) = Mix 40 mg with 10 mL water
      • 10 mM silver nitrate (AgNO3)= Mix 17 mg with 10 mL water - COVER WITH FOIL!
      • 0.1 M ascorbic acid = Mix 0.18 gram with 10 mL water
      • 2.5 M NaCl = Mix 7.3 g NaCl with 50 ml water
  • store 1.5 mL aliquots with each group's sticker at 4ºC


Friday November 13th

  • take out titer plates from Thursday's lab and store at 4ºC


Saturday November 14th

  • take out titer plates from Friday's lab and store at 4ºC


Set up for Day 2 - Tuesday November 17th and Wednesday November 18th

  • quiz!
  • solutions the students made on Day 1 will be used here


Set up for Day 3 - Thursday November 19th and Friday November 20

  • quiz
  • TEM grids
  • copious amounts of ethanol in both 50 mL conicals and squirt bottles
  • enough mortar/pestles for each group (=5)
  • aluminum foil


Monday November 30th

  • put Tuesday lab's mortars into the oven


Tuesday December 1st

  • put Wednesday lab's mortars into the oven


Set up for Day 4 - Tuesday December 1st and Wednesday December 2nd

  • make sure Belcher lab knows we're coming to use their mass balance and space, as well as someone to help with battery assembly
  • stainless steel plate and roller
  • battery demo
  • electrodes massed down in the Belcher lab and then placed in the vacuum oven to dry


Set up for Day 5 - Thursday December 3rd and Friday December 4th

  • working with the Belcher lab to test batteries


Set up for Day 6 - Tuesday December 8th and Wednesday December 9th

  • data from batteries
  • light LED


Other important notes:

  • all antibiotic solutions are stored at 4ºC but powdered tet is in the -20ºC and powdered kan is in the 4ºC
  • taking pictures and posting them on Picasa is really fun! And it gives you a nice visual overview of what you actually did.
  • ER2267 cells are kind of picky and die after some time at 4ºC - streak out a new plate from the stock at -80ºC before beginning the module
  • things that should be kept from module to module:
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