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

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(Procedure)
Line 9: Line 9:
*begin protein expression
*begin protein expression
*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==
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** 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 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.
**Binding and elution buffers were prepared by [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]]
**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 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]].
+
*Tris added to Au/BSA
-
*The following calculations were completed to determine how much tris was needed in each solution. [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]] prepared the tris stock.
+
**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]].
 +
**The following calculations were completed to determine how much tris was needed in each solution. [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]] prepared the tris stock.
**m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
**m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
**1000mM*6mL=1000mM*x
**1000mM*6mL=1000mM*x

Revision as of 23:31, 6 December 2012

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Objectives

  • begin protein expression
  • 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.
    • The following calculations were completed to determine how much tris was needed in each solution. Puja Mody prepared the tris stock.
    • m1v1=m2v2
    • 1000mM*6mL=1000mM*x
      • 6mL Tris stock for 1M solution
    • 500mM*6mL=1000mM*x8
      • 3mL needed for 500mM 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


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