20.109(S19):Prep notes for M3

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20.109(S19): Laboratory Fundamentals of Biological Engineering

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Spring 2019 schedule        FYI        Assignments        Homework        Class data        Communication
       1. Assessing ligand binding        2. Measuring gene expression        3. Engineering biomaterials              


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:

  • incubate for at least 24 hours, or for as long as needed given class schedule
  • 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
    • stir for 12 hours, and for no more than ~14 hours
  • quench biomineralization
    • add 26.7 mL 25 mM Na:PO4 stock solution, resulting in final concentration of Fe and PO4 of 6.45 mM
      • (2.6 g monosodium phosphate monohydrate, 21.8 g disodium phosphate heptahydrate, up to 100 mL in dH2O)
    • stir for 10 minutes
  • check pH
    • adjust to 6.5-7.5 with 50 mM NaOH
    • stir for 6 hours

M3D2

prior to laboratory:

  • prepare aliquots of phage and Na:PO4 for biomineralization demonstration

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