User:Michael F. Nagle/Notebook/Chem 571/2012/09/19: Difference between revisions

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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
 
==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]].
## 1mL .4M IPTG was added to each flask to start protein exprssion
***1M stock solution for tris was made
## Expression cultures were incubated at 160rpm and 37°C for 4 hours.
****1mol/L *.025L = .025mol Tris needed
## 30mL binding buffer was inserted to each flask.
****.025mol * 121.14g/mol = 3.0285g Tris weighed
## Flasks were centrifigued at 4500rpm for 15 minutes
***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.
## Cells were collected and placed in the freezer at -80°C.
****m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
 
****1000mM*6mL=1000mM*x
#Binding buffer
*****6mL Tris stock for 1M solution
##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
****200mM*6mL=1000mM*x
##.02M Tris solution *1L = .02moles Tris needed
*****1.2mL Tris stock for 200mM solution
##.02mol Tris * 121.14g = 2.4228g Tris weighed
****100mM*6mL=1000mM*x
##.5M NaCl *1L =.5mol NaCl needed
*****.6mL Tris stock for 100mM solution
##.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 HAuCl<sub>4</sub> and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M
##m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
##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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__NOTOC__

Revision as of 21:44, 6 December 2012

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Objectives

  • Begin growing E. Coli for ADA expression. The ADA made will be used to nucleate AuNPs
  • make elution and binding buffers to be used for extraction and purification of ADA
  • prepare solutions of tris in Au/BSA, to be analyzed by UV/Vis to determine how various concentrations affect AuNPs in solution

Procedure

  • Expressing ADA
    • Cultures were prepared and E. Coli with a plasmid for Adenosine Deaminase (ADA) were added and incubated overnight, by Mary Mendoza. She also prepared binding and elution buffers for extraction.
    • Binding and elution buffers were prepared by Puja Mody
  • Tris added to Au/BSA
    • In order to determine how various concentrations of tris affect AuNPs in solution, tris was added to 70 [HAuCl4/BSA] solutions prepared last week at .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. These are to be analyzed by UV/Vis.
      • 1M stock solution for tris was made
        • 1mol/L *.025L = .025mol Tris needed
        • .025mol * 121.14g/mol = 3.0285g Tris weighed
      • 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.
        • m1v1=m2v2
        • 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
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