Difference between revisions of "20.109(S18): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (→M2D4) |
Noreen Lyell (Talk | contribs) (→M2D3) |
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*lysis buffer reagents | *lysis buffer reagents | ||
** | ** | ||
| − | *250 μL slurry | + | *250 μL slurry in 2 mL microcentrifuge tube |
*7 mL 1X PBS | *7 mL 1X PBS | ||
*Y μL 1 M MgCl<sub>2</sub> | *Y μL 1 M MgCl<sub>2</sub> | ||
Revision as of 21:47, 31 January 2018
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
per team:
- lysis buffer reagents
- 250 μL slurry in 2 mL microcentrifuge tube
- 7 mL 1X PBS
- Y μL 1 M MgCl2
- 12 μL DNase
- 1.7 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
per team:
- 18 μL 1 mg/mL FKBP12
- buffer reagents
- X μL 1 M Tris-HCL pH=8
- Y μL 100 mM CaCl2
- Z μL chymotrypsin
M2D6
per team:
- 18 μL 1 mg/mL FKBP12
M2D7
per team:
- none
