User:Michael F. Nagle/Notebook/Chem 571/2012/09/19: Difference between revisions
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*make elution and binding buffers | *make elution and binding buffers | ||
*collect E. Coli with ADA | *collect E. Coli with ADA | ||
== | ==Procedure== | ||
#Expressing ADA | #Expressing ADA | ||
##Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth. | ##Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth. | ||
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## Cells were collected and placed in the freezer at -80°C. | ## 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 | ##.02M Tris solution *1L = .02moles Tris | ||
##.02mol Tris * 121.14g = 2.4228g Tris | ##.02mol Tris * 121.14g = 2.4228g Tris | ||
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##.5M imidazole *.5L = .25mol imidazole | ##.5M imidazole *.5L = .25mol imidazole | ||
##.25mol*68.077g/mol=17.019g imidazole | ##.25mol*68.077g/mol=17.019g imidazole | ||
#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 HAuCl<sub>4</sub> and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M | #Tris buffer stock solution was inserted into HAuCl<sub>4</sub> and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M |
Revision as of 12:00, 1 October 2012
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Objectives
Procedure
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