Difference between revisions of "20.109(S20): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (→M1D6) |
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==M1D4== | ==M1D4== | ||
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'''per team:''' | '''per team:''' | ||
+ | *12 mL TBS-T | ||
+ | *4 mL TBS | ||
+ | *front laboratory bench: dH<sub>2</sub> reservoirs for 'dunking' slides | ||
==M1D5== | ==M1D5== |
Revision as of 20:32, 5 February 2020
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
- at the 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: dH2 reservoirs for 'dunking' slides
M1D5
prior to laboratory:
per team:
during the laboratory:
M1D6
prior to laboratory:
per team:
during the laboratory:
Bring to Koehler lab
M1D7
per team:
- none