20.109(S20): Prep notes for M1

M1D1

per team:

• 25 μL pET_MBP_SNAP_TDP43-RRM12 (25 ng/uL)
• 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
• inoculate BL21-A1 pET_MBP_SNAP_TDP43-RRM12 and induce protein expression:
  • inoculate 5 mL LB containing kanamycin with BL21-A1 pET_MBP_SNAP_TDP43-RRM12 and culture overnight
  • dilute 1:10 in 50 mL LB containing kanamycin and culture ~4 hours (until log phase)
  • add IPTG (1 mM) and arabinose (0.2%) and incubate 2.5 hr at 37 °C
  • pellet cells at 3000g for 10 min.
  • also prepare uninduced pellets

per team:

• 25 μL 6x loading dye
• 3 mL lysis buffer: 50 mM NaH₂PO₄, 300 mM NaCl
  • students to add imidazole to 10 mM and 5 μL lysonase
• 15 μL 2.5 M imidazole
• 500 μL 50% nickel-resin (Ni-NTA) slurry
• 1.2 mL 1X PBS
• 8 mL 10 mM imidazole in 1X PBS
• 3 mL HRV 3C buffer

• front laboratory bench: lysonase, SnapTag substrate, DTT, HRV 3C protease

M1D3

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

M1D4

per team:

• 12 mL TBS-T
• 4 mL TBS

• front laboratory bench: dH₂O reservoirs for 'dunking' slides

M1D5

per team:

• none

M1D6

per team:

• none

M1D7

per team:

• none