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

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==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 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]]
#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.
*In order to determine how various concentrations of tris affect AuNPs in solution, tris solution 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. They 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>
##500mM*6mL=1000mM*x8##3mL
##500mM*6mL=1000mM*x8##3mL

Revision as of 21:26, 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

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
  • In order to determine how various concentrations of tris affect AuNPs in solution, tris solution was added to 70 [HAuCl4/BSA] solutions prepared last week at .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. They 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.
    1. m1v1=m2v2
    2. 500mM*6mL=1000mM*x8##3mL
    3. 1000mM*6mL=1000mM*x
    4. 6mL Tris stock for 1M solution
    5. 200mM*6mL=1000mM*x
    6. 1.2mL Tris stock for 200mM solution
    7. 100mM*6mL=1000mM*x
    8. .6mL Tris stock for 100mM solution
    9. 50mM*6mL=1000mM*x
    10. .3mL Tris stock for 50mM solution
    11. 5mM*6mL=1000mM*x
    12. .03mL Tris stock for 5mM solution
    13. .5mM*6mL=1000mM*x
    14. .003mL Tris stock for .5mM solution
    15. .05mM*6mL=1000mM*x
    16. .0003mL Tris stock for .05mM solution
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