Difference between revisions of "20.109(F15): TA's notes for module 3"

Revision as of 02:03, 10 December 2015

Day 1

The week preceding the lab:

• Make LB:
  • 10 g tryptone
  • 5 g yeast extract
  • 10 g NaCl
  • 1 L deionized water
  • autoclave 30 minutes

On the instructors' bench:

• 50 mL conical tubes
• 25 mL pipets
• a couple of motorized pipet aids
• 2 mL eppendorf tubes
• TBS: 500 mL
• PEG/NaCl: 500 mL
• deionized water: 50 mL
• quartz cuvettes

On the students' bench:

• bucket of ice
• 2 x 40 mL overnight culture of XL1-Blue infected with M13 phage
• 250 mL erlenmeyer flask

By the scale:

• (NH₄)₂Fe(SO₄)₂
• stirbars

In 4 °C fridge:

• orbital shaker (speed 120)

Day 2

Between Day 1 and Day 2:

• 1 M pH 7.5 Na:PO₄ solution:
  • 2.6% Monosodium phosphate, monohydrate - 2.6 g per 100 mL
  • 21.8% Disodium phosphate, heptahydrate - 21.8 g per 100 mL
  • These are calculated values that will result in a 100 mL solution of pH 7.5

• Aliquot ~ 10 mL of students phage-Fe incubating mixture and transfer to 20 mL glass vial. This will be used for demonstration purposes for their next lab section
• 12-16 hours after phage-Fe incubation, add 1 mL of Na:PO₄ solution to each groups flask (result is a 10 mM phosphate solution)

On the instructors' bench:

• 50 mL conical tubes
• 15 mL conical tubes
• tweezers (2 per team)
• TEM grids for practice (some)
• new TEM grids (2 per team)
• sign-up sheet for location of samples in TEM holder
• 100% ethanol (20 μL per team, so a couple of 1.5 mL eppendorf tubes suffice)

On students' benches:

• For demo purposes:
  • ~10 mL of each students phage-Fe mixture which was saved previously
  • 1 mL aliquot of 1 M pH 7.5 Na:PO₄ solution in eppendorf
  • Allow students to add 100 μL of phosphate solution to their phage-Fe mixture
  • Solution should turn green-ish brown. The darker color, compared to their overnight phage-Fe+phosphate mixture may be due to the prolonged phage-Fe incubation (which lasted over the weekend), which may have formed iron oxide particles (FeO or Fe₂O₃) that are more opaque and rusty colored.
• Returned 90 mL solution of phage-Fe+phosphate solution which was completed between Day 1 & 2 by instructors.

Note:

• An instructor should go around with the grid holder, and allow each group to take two tweezers and grab two Cu-TEM grids
• Grids should be held by the tweezers at all times during depositing and washing steps.
• Place some kimwipes beneath the tweezers, and set tweezers with grid on the bench.

Day 3

Materials

• Flat stainless steel plate (INSERT CATALOG #)
• Stainless steel spatulas
• Smooth stainless steel roller (INSERT CATALOG #)
• Pestle and Mortar (INSERT CATALOG #)

Instructor materials

• 3x 3/8 puncher
• superP carbon powder
• Teflon 8A (aka. binder)

Flowthrough

• Ten groups were successfully handled between 2 TAs and 1 Instructor. The groups were split between rooms 76-589 and 76-591 of the Belcher lab. Each room has its own weigher and stock of superP and binder.
• Due to limited weighers, groups have to take turns weighing out their dried active material.
  • Weights should be taken on a triangle cut weigh paper. Once weights are calculated, active material can be placed directly into the pestle
• Each group weighs out a calculated amount of superP and mixes it with their active material in the pestle.
• Team members should split up
  • One should continue grinding their material. When complete (ask instructor/TA), gently transfer onto steel plate. DO NOT ROLL
  • Another student should weigh out binder. Careful: small amounts (10-50 mg) of binder needs to be weighed, an error of +/- 10% is OK.
  • Slowly and carefully transfer binder on its weigh paper and sprinkle on top of the mixed active material and superP
• Go crazy and start rolling
• Ask instructors for help or input if film is breaking, not shiny, or is difficult to punch out with the 3/8 puncher.
• All students electrodes should be weighed out and transferred gently into one well of a 6 well plate. The cover over each well should be clearly labeled with lab time and group color.
• TA: when a plate has an electrode in each well, put on cover and tape each corner. Transfer the plate into the 80^OC vacuum oven.

Cleanup

• Stainless steel plates, spatulas, and rollers can be cleaned with thorough washes of water to remove residual electrode materials.
• Cleaning the Pestle and Mortar is difficult. Clean with alternating washes of water and alconox. Many residual material may remain on the pestle, this is unavoidable. Pestle and Mortar can be further heated at > 8O^OC overnight to etch away material that can be later washed away.

Day 4

Day 5