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
• 525 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 &um;M chymotrypsin in 1 mM HCl containing 2 mM CaCl₂ to 2 mM chymotrypsin in H₂O (1:100)

per team:

• 18 μL 1 mg/mL FKBP12
• buffer reagents
  • 1 mL 1 M Tris-HCl, pH=8
  • 50 μL 2 μM chymotrypsin

during the laboratory:

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

M2D6

per team:

• 18 μL 1 mg/mL FKBP12

M2D7

per team:

• none