Difference between revisions of "20.109(S18): Prep notes for M1"
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Noreen Lyell (Talk | contribs) (→M2D5) |
Noreen Lyell (Talk | contribs) |
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'''per team:''' | '''per team:''' | ||
− | * | + | *25 μL 6x loading dye |
− | * | + | *525 μL 1 M IPTG |
==M2D3== | ==M2D3== | ||
Line 25: | Line 25: | ||
*lysis buffer reagents | *lysis buffer reagents | ||
− | ** | + | **170 μL 1 M Tris, pH = 7 |
− | *250 μL slurry in 2 mL microcentrifuge tube | + | **470 μL 1 M NaCl |
+ | **770 μL 40% glycerol | ||
+ | **5 μL 1 M DTT | ||
+ | **35 μL 10 mM AEBSF | ||
+ | **1.8 mL sterile H<sub>2</sub>O | ||
+ | *250 μL slurry (Ni-NTA resin) in 2 mL microcentrifuge tube | ||
*2.5 mL 1X PBS | *2.5 mL 1X PBS | ||
− | * | + | *40 μL 1 M MgCl<sub>2</sub> |
*12 μL DNase | *12 μL DNase | ||
*2 mL 1X PBS containing 10 mM imidazole | *2 mL 1X PBS containing 10 mM imidazole |
Revision as of 16:57, 3 February 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:
- 25 μL 6x loading dye
- 525 μL 1 M IPTG
M2D3
prior to laboratory:
- prepare dialysis buffer, X mL per team
per team:
- lysis buffer reagents
- 170 μL 1 M Tris, pH = 7
- 470 μL 1 M NaCl
- 770 μL 40% glycerol
- 5 μL 1 M DTT
- 35 μL 10 mM AEBSF
- 1.8 mL sterile H2O
- 250 μL slurry (Ni-NTA resin) in 2 mL microcentrifuge tube
- 2.5 mL 1X PBS
- 40 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
during 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