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| ==Objectives== | | ==Objectives== |
| *begin protein expression | | *Begin growing <i>E. Coli</i> for ADA expression. The ADA made [[User:Michael_F._Nagle/Notebook/Chem_571/2012/11/28|will be used to nucleate AuNPs]] |
| *make elution and binding buffers | | *make elution and binding buffers to be used for extraction and purification of ADA |
| *collect E. Coli with ADA | | *prepare solutions of tris in Au/BSA, to be analyzed by UV/Vis to determine how various concentrations affect AuNPs in solution |
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| ==Procedure== | | ==Procedure== |
| #Expressing ADA
| | *Expressing ADA |
| ## Procedure for protein expression was followed with noted changes, and can be found here: [[AU_Biomaterials_Design_Lab:Protocols/Protein_Expression]]
| | ** Cultures were prepared and <i>E. Coli</i> with a plasmid for Adenosine Deaminase (ADA) were added and incubated overnight, by [[User:Mary_Mendoza/Notebook/CHEM_571_Experimental_Biological_Chemistry_I/2012/09/19|Mary Mendoza]]. She also prepared binding and elution buffers for extraction. |
| ## Tris stock for 1M solution
| | **Binding and elution buffers were prepared by [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]] |
| Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
| | *Tris added to Au/BSA |
| ## To inoculate the expression culture, resuspended cells were divided between flasks.
| | **In order to determine how various concentrations of tris affect AuNPs in solution, tris was added to 70 [HAuCl<sub>4</sub>/BSA] solutions prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/12|last week]] at .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. These [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/25|are to be analyzed by UV/Vis]]. |
| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''': more than that was done. those cells were then added to the 1L fernbach flasks. what time did this all occur? you need to link ot Mary's notebook since she is the one who actually did the experimental procedure.
| | ***1M stock solution for tris was made |
| ## 1mL .4M IPTG was added to each flask to start protein exprssion
| | ****1mol/L *.025L = .025mol Tris needed |
| ## Expression cultures were incubated at 160rpm and 37°C for 4 hours.
| | ****.025mol * 121.14g/mol = 3.0285g Tris weighed |
| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':be more precise. at exactly what time was hte IPTG added? what time were hte expression cultures removed? | | ***The following calculations were completed to determine how much tris was needed in each solution for concentrations 1M, 500mM, 200mM and 100mM. A serial dilution was done to prepare concentrations .05mM-50mM from the 500mM solution. |
| ## 30mL binding buffer was inserted to each flask.
| | ****m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub> |
| ## Flasks were centrifigued at 4500rpm for 15 minutes
| | ****1000mM*6mL=1000mM*x |
| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':binding buffer was used to resuspend cells after centrifugation. you need to link to someone's notebook if you did not do it. | | *****6mL Tris stock for 1M solution |
| ## Cells were collected and placed in the freezer at -80°C.
| | ****200mM*6mL=1000mM*x |
| | | *****1.2mL Tris stock for 200mM solution |
| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':you need more details.
| | ****100mM*6mL=1000mM*x |
| | | *****.6mL Tris stock for 100mM solution |
| #Binding buffer
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| ##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
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| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':no made with...those are the exact chemicals and concentrations of the buffer.
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| ##.02M Tris solution *1L = .02moles Tris needed
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| ##.02mol Tris * 121.14g = 2.4228g Tris weighed
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| ##.5M NaCl *1L =.5mol NaCl needed
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| ##.5mol*58.439=29.2195g NaCl
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| ##29.2147gNaCl weighed
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| ##30mM imidazole *1L = .03mol imidazole needed
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| ##.03mol*68.077g/mol=2.0423g imidazole weighed
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| ##HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
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| #A buffer for elution was made with 20mM Tris, .5M NaCl and 500mM amidazole
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| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':see above comment | |
| ##.02M Tris solution *.5L = .01moles Tris needed
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| ##.01mol Tris * 121.14g = 1.2114g Tris
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| ##1.2164g Tris weighed
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| ##.5M NaCl *.5 = .25mol NaCl needed
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| ##.25mol*58.439g/mol=14.6098gNaCl weighed
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| ##.5M imidazole *.5L = .25mol imidazole needed
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| ##.25mol*68.077g/mol=17.019g imidazole weighed
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| #HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
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| #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
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| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':terminology - inserted is not the appropriate term. you add to solutions or reactions because it leads to an increase in moles or volume, not insert.
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| ##m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
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| ##500mM*6mL=1000mM*x8##3mL
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| ##1000mM*6mL=1000mM*x
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| ##6mL Tris stock for 1M solution
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| ##200mM*6mL=1000mM*x
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| ##1.2mL Tris stock for 200mM solution
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| ##100mM*6mL=1000mM*x
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| ##.6mL Tris stock for 100mM solution
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| ##50mM*6mL=1000mM*x
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| ##.3mL Tris stock for 50mM solution
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| ##5mM*6mL=1000mM*x
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| ##.03mL Tris stock for 5mM solution
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| ##.5mM*6mL=1000mM*x
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| ##.003mL Tris stock for .5mM solution
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| ##.05mM*6mL=1000mM*x
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| ##.0003mL Tris stock for .05mM solution
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| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:34, 7 October 2012 (EDT)''':why are you making these buffers? how much as added to what concentrations of HAuCl4 nad BSA? is this before or after AuNP formation?
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| ==Observations==
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| One of the flasks with broth appeared a different shade of yellow this morning prior to centrifuging.
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| *'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:34, 7 October 2012 (EDT)''':why is this important? did this change your procedure?
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