Objectives
- begin protein expression
- make elution and binding buffers
- collect E. Coli with ADA
Procedure
- Expressing ADA
- Procedure for protein expression was followed with noted changes, and can be found here: AU_Biomaterials_Design_Lab:Protocols/Protein_Expression
- Tris stock for 1M solution
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.
- Binding buffer
- made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
- .02M Tris solution *1L = .02moles Tris needed
- .02mol Tris * 121.14g = 2.4228g Tris weighed
- .5M NaCl *1L =.5mol NaCl needed
- .5mol*58.439=29.2195g NaCl
- 29.2147gNaCl weighed
- 30mM imidazole *1L = .03mol imidazole needed
- .03mol*68.077g/mol=2.0423g imidazole 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 needed
- .01mol Tris * 121.14g = 1.2114g Tris
- 1.2164g Tris weighed
- .5M NaCl *.5 = .25mol NaCl needed
- .25mol*58.439g/mol=14.6098gNaCl weighed
- .5M imidazole *.5L = .25mol imidazole needed
- .25mol*68.077g/mol=17.019g imidazole weighed
- HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
- 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 Tris stock for 1M solution
- 200mM*6mL=1000mM*x
- 1.2mL Tris stock for 200mM solution
- 100mM*6mL=1000mM*x
- .6mL Tris stock for 100mM solution
- 50mM*6mL=1000mM*x
- .3mL Tris stock for 50mM solution
- 5mM*6mL=1000mM*x
- .03mL Tris stock for 5mM solution
- .5mM*6mL=1000mM*x
- .003mL Tris stock for .5mM solution
- .05mM*6mL=1000mM*x
- .0003mL Tris stock for .05mM solution
Observations
One of the flasks with broth appeared a different shade of yellow this morning prior to centrifuging.
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