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

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(Objectives)
(Procedure)
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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 to be used for extraction and purification of ADA
 
*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==
 
==Procedure==
#Expressing ADA
+
*Expressing ADA
## Cultures were prepared and E. Coli 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.
+
** 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.
 
+
**Binding and elution buffers were prepared by [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]]
#In order to determine how various concentrations of Tris affect the formation of AuNP by BSA, tris buffer solution was added to HAuCl<sub>4</sub> and BSA solutions prepared [[User:Michael_F._Nagle/Notebook/Chem_571/2012/09/12|last week]] at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. They are to be analyzed by UV/Vis.
+
*Tris added to Au/BSA
##m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
+
**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]].
##500mM*6mL=1000mM*x8##3mL
+
***1M stock solution for tris was made
##1000mM*6mL=1000mM*x
+
****1mol/L *.025L = .025mol Tris needed
##6mL Tris stock for 1M solution
+
****.025mol * 121.14g/mol = 3.0285g Tris weighed
##200mM*6mL=1000mM*x
+
***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.
##1.2mL Tris stock for 200mM solution
+
****m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
##100mM*6mL=1000mM*x
+
****1000mM*6mL=1000mM*x
##.6mL Tris stock for 100mM solution
+
*****6mL Tris stock for 1M solution
##50mM*6mL=1000mM*x
+
****200mM*6mL=1000mM*x
##.3mL Tris stock for 50mM solution
+
*****1.2mL Tris stock for 200mM solution
##5mM*6mL=1000mM*x
+
****100mM*6mL=1000mM*x
##.03mL Tris stock for 5mM solution
+
*****.6mL Tris stock for 100mM solution
##.5mM*6mL=1000mM*x
 
##.003mL Tris stock for .5mM solution
 
##.05mM*6mL=1000mM*x
 
##.0003mL Tris stock for .05mM solution
 
 
 
 
__NOTOC__
 
__NOTOC__

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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