20.109(F15): TA's notes for module 3

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.

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.