20.109(S19): Prep notes for M1

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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              


M1D1

per team:

  • 200 μL nuclease-free H2O

during the laboratory:

  • restriction enzymes and buffers
  • filtered pipet tips

M1D2

prior to laboratory:

  • prepare 1% agarose gels, 1 per 2 teams
  • subculture BL21(DE3) pRSETb_FKBP12 1:10 into 50 mL LB containing 100μg / mL amp and 34 μg / mL cam, 1 per team

per team:

  • 25 μL 6x loading dye
  • 520 μL 0.1 M IPTG

M1D3

prior to laboratory:

  • prepare dialysis buffer (1X PBS), 1 L per team

per team:

  • 15 μL 50 mg/mL lysozyme
  • lysis buffer reagents
    • 170 μL 1 M Tris, pH = 7
    • 470 μL 1 M NaCl
    • 770 μL 40% glycerol
    • 5 μL 1 M DTT
    • 35 μL 10 mM AEBSF
    • 1.8 mL sterile H2O
  • 250 μL slurry (Ni-NTA resin) in 2 mL microcentrifuge tube
  • 2.5 mL 1X PBS
  • 40 μL 1 M MgCl2
  • 12 μL DNase
  • 5 mL 1X PBS containing 10 mM imidazole
  • 3.5 mL 1X PBS containing 250 mM imidazole


2/12/18 Calculations for 10 aliquots of Imidazole, make fresh the day of:

  • 250mM Imidazole: 0.68 g of Imidazole in 40 mL 1X PBS
  • 10mM Imidazole: 2 mL of 250mM stock + 48 mL 1X PBS

M1D4

prior to laboratory:

  • boil stained and unstained molecular weight standards, if required
  • prepare electrophoresis buffer, 1 chamber volume per 2 teams

per team:

  • 20 μL Laemmli buffer
  • 50 mL coomassie stain
  • BSA reagents
    • 110 μL albumin
    • 1.3 mL 1X PBS
    • 17 mL BCA Reagent A
    • 400 μL BCA Reagent B

M1D5

prior to laboratory:

  • dilute 200 μM chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl2) to 2 μM chymotrypsin in H2O
  • prepare reagents
    • DMSO: 40% in H2O
    • rapamycin: 20 μM in 40% DMSO
    • ligands: 8 mM in 40% DMSO

per team:

  • 18 μL 1 mg/mL FKBP12
  • buffer reagents
    • 1.3 mL 1 M Tris-HCl, pH = 8
    • 65 μL 2 μM chymotrypsin
    • 5 mL H2O
  • 20 μL 40% DMSO; prepared such that 1 μL needed per reaction (final concentration = 0.2% in 200 μL)
  • 10 μL 20 μM rapamycin; prepared such that 1 μL needed per reaction (final concentration = 10 μM in 200 μL)
  • 10 μL 8 mM Chembridge ligands (most stocks at 100 mM); prepared such that 1 μL needed per reaction (final concentration = 40 μM in 200 μL)

during the laboratory:

  • prepare 5 mM suc-AAFP-pNA in TFE containing 460 mM LiCl

M1D6

prior to laboratory:

  • prepare serial dilutions of reagents (MIX WELL BETWEEN DILUTIONS)
    • dye: 1000x → 100x (3 μL dye + 27 μL 1X PBS); 100x → 5x (30 μL of 100x dye + 570 μL 1X PBS)
    • DMSO: 100% → 1% (5 μL DMSO + 495 μL 1X PBS)
    • rapamycin: prepare 5 mM in DMSO; 5 mM → 500 μM (5 μL of 5 mM rapamycin + 45 μL 1X PBS); 500 μM → 50 μM (10 μL of 500 μM rapamycin + 90 μL 1X PBS)
    • ligands: 100 mM → 20 mM (1 μL ligand in 4 μL DMSO); 20 mM → 2 mM (5 μL of 10 mM ligand + 45 μL 1X PBS); 2 mM → 200 μM (5 μL of 1 mM ligand + 45 μL 1X PBS)

per team:

  • 18 μL 1 mg/mL FKBP12
  • 80 μL 5x dye solution in 1X PBS (includes enough for fkbp and control protein wells)
  • 24 μL 1% DMSO in 1X PBS
  • 10 μL 50 μM rapamycin in 1X PBS
  • 20 μL 200 μM ligand in 1X PBS
  • 50 μL 1X PBS
  • 35 μL Control buffer
  • 55 μL sterile water


during the laboratory:

  • control protein
  • control ligand
  • sterile H2O
  • filtered pipet tips

Bring to Koehler lab

  • control protein (2 ul/well, 6.5 uL/mastermix)
  • FKBP12 (ab85840, 1.2uL/well, 3.9 uL/mastermix)
  • plates & seals
  • Pipettes & tips

M1D7

per team:

  • none