20.109(S19): Prep notes for M1

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