20.109(S18):Prep notes for M3

M3D1

prior to laboratory:

• prepare LB (10 g tryptone, 5 g yeast extract, 10 g NaCl, up to 1 L in dH₂O)

per team:

• 2 x 40 mL overnight cultures of M13-infected XL1-Blue cells
• 30 mL 20% PEG-8000/2.5 M NaCl
• 50 mL deionized water

during the laboratory:

• 50 mL conical tubes
• 25 mL pipets
• 2 mL eppendorf tubes
• quartz cuvettes
• 250 mL erlenmeyer flasks
• ice buckets
• stirbars

after the laboratory:

• add Fe2+
  • add 26.7 mL of 25 mM (NH₄)2Fe(SO₄)₂ added to 50 mL phage-AuNP, resulting in a final concentration of 8.7 mM
• aliquot for in-class demonstration
  • transfer 1 mL of student's phage-Fe to 2 mL tube
• quench biomineralization
  • add 26.7 mL 25 mM Na:PO4 stock solution, resulting in final concentration of Fe and PO₄ of 6.45 mM
  • stir for 10 minutes
• check pH
  • adjust to 6.5-7.5 with 50 mM LiOH (or NaOH, NH3.H2O)
  • stir for 6 hours

M3D2

prior to laboratory:

• 1 M Na:PO₄, pH = 7.5 (2.6 g monosodium phosphate monohydrate, 21.8 g disodium phosphate heptahydrate, up to 100 mL in dH₂O)

per team:

• 1 mL aliquots of student's phage in 2 mL tubes
• 25 μL 1 M Na:PO₄, pH = 7.5
• 50 μL 100% ethanol

during the laboratory:

• 50 mL conical tubes
• 15 mL conical tubes
• tweezers
• old TEM grids, for practice
• new TEM grids, 2 per team
• sign-up sheet for location of samples in TEM case

M3D3

during the laboratory:

• flat stainless steel plates
• smooth stainless steel roller
• stainless steel spatulas
• mortar and pestle
• 3/8 puncher
• superP carbon powder
• Teflon 8A, binder

M3D4

per team:

• none

M3D5

per team:

• none