Difference between revisions of "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:

• X μL 6x loading dye
• Y μL 1 M IPTG

M2D3

prior to laboratory:

• prepare dialysis buffer, X mL per team

per team:

• lysis buffer reagents
• 250 μL slurry in 2 mL microcentrifuge tube
• 2.5 mL 1X PBS
• Y 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
  • 1300 μL 1X PBS
  • X μL BCA Reagent A
  • Y μL BCA Reagent B

M2D5

prior to laboratory:

• prepare 0.2 mM chymotrypsin in 1 mM HCl containing 2 mM CaCl₂

per team:

• 18 μL 1 mg/mL FKBP12
• buffer reagents
  • X μL 1 M Tris-HCL pH=8
  • Z μL 0.2 mM chymotrypsin

after 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