Difference between revisions of "20.109(S18): Prep notes for M1"
From Course Wiki
Noreen Lyell (Talk | contribs) (→M2D5) |
Noreen Lyell (Talk | contribs) (→M2D5) |
||
Line 63: | Line 63: | ||
*18 μL 1 mg/mL FKBP12 | *18 μL 1 mg/mL FKBP12 | ||
*buffer reagents | *buffer reagents | ||
− | **1 mL 1 M Tris- | + | **1 mL 1 M Tris-HCl, pH=8 |
**50 μL 2 μM chymotrypsin | **50 μL 2 μM chymotrypsin | ||
Revision as of 17:01, 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 0.1 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
- 1.3 mL 1X PBS
- 17 mL BCA Reagent A
- 400 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
- 1 mL 1 M Tris-HCl, pH=8
- 50 μL 2 μM 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