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

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(Objectives)
Current revision (23:44, 6 December 2012) (view source)
(Procedure)
 
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==Objectives==
==Objectives==
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*begin protein expression
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*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
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*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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#Expressing ADA
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*Expressing ADA
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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.
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** 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.
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**Binding and elution buffers were prepared by [[User:Puja_Mody/Notebook/Chem_571:_Gold_Nanoparticles/2012/09/19|Puja Mody]]
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#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.
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*Tris added to Au/BSA
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##m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
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**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]].
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##500mM*6mL=1000mM*x8##3mL
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***1M stock solution for tris was made
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##1000mM*6mL=1000mM*x
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****1mol/L *.025L = .025mol Tris needed
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##6mL Tris stock for 1M solution
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****.025mol * 121.14g/mol = 3.0285g Tris weighed
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##200mM*6mL=1000mM*x
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***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.
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##1.2mL Tris stock for 200mM solution
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****m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
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##100mM*6mL=1000mM*x
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****1000mM*6mL=1000mM*x
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##.6mL Tris stock for 100mM solution
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*****6mL Tris stock for 1M solution
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##50mM*6mL=1000mM*x
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****200mM*6mL=1000mM*x
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##.3mL Tris stock for 50mM solution
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*****1.2mL Tris stock for 200mM solution
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##5mM*6mL=1000mM*x
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****100mM*6mL=1000mM*x
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##.03mL Tris stock for 5mM solution
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*****.6mL Tris stock for 100mM 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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__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
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