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]]
## 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.


*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':you need more details.


#Binding buffer
#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 tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. They are to be analyzed by UV/Vis.
##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':no made with...those are the exact chemicals and concentrations of the buffer.
##.02M Tris solution *1L = .02moles Tris needed
##.02mol Tris * 121.14g = 2.4228g Tris weighed
##.5M NaCl *1L =.5mol NaCl needed
##.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
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':see above comment
##.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
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':terminology - inserted is not the appropriate term. you add to solutions or reactions because it leads to an increase in moles or volume, not insert.
##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 20:13, 25 October 2012

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Objectives

  • begin protein expression
  • make elution and binding buffers
  • collect E. Coli with ADA

Procedure

  1. Expressing ADA
    1. 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.


  1. In order to determine how various concentrations of Tris affect the formation of AuNP by BSA, tris buffer solution was added to HAuCl4 and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M. They are to be analyzed by UV/Vis.
    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
  • Abigail E. Miller 11:34, 7 October 2012 (EDT):why are you making these buffers? how much as added to what concentrations of HAuCl4 nad BSA? is this before or after AuNP formation?

Observations

One of the flasks with broth appeared a different shade of yellow this morning prior to centrifuging.

  • Abigail E. Miller 11:34, 7 October 2012 (EDT):why is this important? did this change your procedure?


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