Objectives
- begin protein expression
- make elution and binding buffers
- collect E. Coli with ADA
Entry title
- Expressing ADA
- Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
- To inoculate the expression culture, resuspended cells were divided between flasks.
- 1mL .4M IPTG was added to each flask to start protein exprssion
- Expression cultures were incubated at 160rpm and 37°C for 4 hours.
- 30mL binding buffer was inserted to each flask.
- Flasks were centrifigued at 4500rpm for 15 minutes
- Cells were collected and placed in the freezer at -80°C.
- Elution Buffer
- .02M Tris solution was needed for 10mL buffer.
- .02M Tris * .01L = .0002moles/liter
- .0002mol *121.14= .02423g Tris
- .249 was the actual amount weighed and put in 7mL water.
- HCl and NaOH were added dropwise until the pH was 7.53, between a target range of 7-8.
- .4g IPTG was weighed and placed in 4mL of buffer
1mL of IPTG solution was added to each expression culture.
- Buffer for binding was made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
- .02M Tris solution *1L = .02moles Tris
- .02mol Tris * 121.14g = 2.4228g Tris
- .5M NaCl *1L =.5mol
- .5mol*58.439=29.2195gNaCl
- 29.2147gNaCl weighed
- 30mM imidazole *1L = .03mol imidazole
- .03mol*68.077g/mol=2.0423g imidazole
- 2.4721g Tris weighed
- HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
- A buffer for elution was made with 20mM Tris, .5M NaCl and 500mM amidazole
- .02M Tris solution *.5L = .01moles Tris
- .01mol Tris * 121.14g = 1.2114g Tris
- 1.2164g Tris weighed
- .5M NaCl *.5 = .25mol
- .25mol*58.439g/mol=14.6098gNaCl
- .5M imidazole *.5L = .25mol imidazole
- .25mol*68.077g/mol=17.019g imidazole
- Tris buffer stock solution was inserted into HAuCl4 and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M
- m1v1=m2v2
- 500mM*6mL=1000mM*x8##3mL
- 1000mM*6mL=1000mM*x
- 6mL
- 200mM*6mL=1000mM*x
- 1.2mL
- 100mM*6mL=1000mM*x
- .6mL
- 50mM*6mL=1000mM*x
- .3mL
- 5mM*6mL=1000mM*x
- .03mL
- .5mM*6mL=1000mM*x
- .003mL
- .05mM*6mL=1000mM*x
- .0003mL
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