20.109(S18): Prep notes for M1

M1D1

• 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

M2D3

prior to laboratory:

• prepare dialysis buffer, X mL per team

per team:

• 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
• 2 mL 1X PBS containing 10 mM imidazole
• 3.5 mL 1X PBS containing 250 mM imidazole

M2D4

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

M2D5

prior to laboratory:

• dilute 200 μM chymotrypsin (prepared in 1 mM HCl containing 2 mM CaCl₂) to 2 μM chymotrypsin in H₂O

per team:

• 18 μL 1 mg/mL FKBP12
• buffer reagents
  • 0.8 mL 1 M Tris-HCl, pH=8 containing
  • 40 μL 2 μM chymotrypsin
• X μL DMSO (?), prepare such that 1 μL needed per reaction (final concentration = 0.2% in 200 μL)
• Y μL B μM rapamycin (?), prepare such that 1 μL needed per reaction (final concentration = 10 μM in 200 μL)
• Chembridge ligands (most at 100 mM), prepare such that 1 μL needed per reaction (final concentration = 40 μM in 200 μL)

aliquot amounts calculated for each team only using Abcam protein and 15 reactions, if in-house protein used aliquots should be increased and/or if ligands not used values should be reduced

during the laboratory:

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

M2D6

prior to laboratory:

• prepare serial dilutions of all reagents
  • dye: 1000x → 100x (2uL dye + 18uL PBS), 100x-> 5x (10uL of 100x dye + 190uL PBS)

B. Rapamycin –conc in well will be 10uM Make 5mM solution in DMSO 4.57mg/ml Dilute 5mM→ 0.5mM (or 500uM) in PBS (5uL of 5mM solution +45uL PBS) MIX WELL Dilute 500uM → 50uM in PBS 50uM will be the stock conc used in the reaction C. ChemBridge compounds (most are at 100mM) -conc in well will be 20uM 100mM→10mM in DMSO (1uL compound +9uL DMSO) 10mM→ 1mM into PBS (5uL of 10mM stock +45uL PBS) MIX WELL 1mM→ 0.1mM (100uM) in PBS 100uM is the stock concentration used in the reaction D. DMSO control (final conc 0.2%) add 1uL DMSO to 9uL PBS (mix well) (10%) Dilute 10%-->1% (this is used for the reaction)

per team:

• 18 μL 1 mg/mL FKBP12
• X μL 5x dye solution in 1X PBS
• Y μL 1% DMSO in 1X PBS
• Z μL 50 μM rapamycin in 1X PBS
• A μL 100 μM ligand in 1X PBS

M2D7

per team:

• none