Difference between revisions of "20.109(S20): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (→M1D2) |
Noreen Lyell (Talk | contribs) (→M1D2) |
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*25 μL 6x loading dye | *25 μL 6x loading dye | ||
− | *3 mL lysis buffer: 50 mM NaH<sub>2</sub>PO<sub>4</sub>, 300 mM NaCl | + | *3 mL lysis buffer: 50 mM NaH<sub>2</sub>PO<sub>4</sub>, 300 mM NaCl |
**students to add imidazole to 10 mM and 5 μL lysonase | **students to add imidazole to 10 mM and 5 μL lysonase | ||
*15 μL 2.5 M imidazole | *15 μL 2.5 M imidazole | ||
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*3 mL HRV 3C buffer | *3 mL HRV 3C buffer | ||
− | * | + | |
+ | *front laboratory bench: lysonase, SnapTag substrate, DTT, HRV 3C protease | ||
==M1D3== | ==M1D3== | ||
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'''prior to laboratory:''' | '''prior to laboratory:''' | ||
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**400 μL BCA Reagent B | **400 μL BCA Reagent B | ||
− | == | + | ==M1D4== |
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− | * | + | *12 mL TBS-T |
− | * | + | *4 mL TBS |
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− | * | + | *front laboratory bench: dH<sub>2</sub>O reservoirs for 'dunking' slides |
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− | ''' | + | ==M1D5== |
+ | '''per team:''' | ||
− | * | + | *none |
==M1D6== | ==M1D6== | ||
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==M1D7== | ==M1D7== |
Latest revision as of 20:35, 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
- 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