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

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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
*make elution and binding buffers to be used for extraction and purification of ADA
*collect E. Coli with 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]]
** 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]]
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':you need more details.
*Tris added to Au/BSA
 
**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]].
#Binding buffer
***1M stock solution for tris was made
##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
****1mol/L *.025L = .025mol Tris needed
*'''[[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.  
****.025mol * 121.14g/mol = 3.0285g Tris weighed
##.02M Tris solution *1L = .02moles Tris needed
***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.
##.02mol Tris * 121.14g = 2.4228g Tris weighed
****m<sub>1</sub>v<sub>1</sub>=m<sub>2</sub>v<sub>2</sub>
##.5M NaCl *1L =.5mol NaCl needed
****1000mM*6mL=1000mM*x
##.5mol*58.439=29.2195g NaCl
*****6mL Tris stock for 1M solution
##29.2147gNaCl weighed
****200mM*6mL=1000mM*x
##30mM imidazole *1L = .03mol imidazole needed
*****1.2mL Tris stock for 200mM solution
##.03mol*68.077g/mol=2.0423g imidazole weighed
****100mM*6mL=1000mM*x
##HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
*****.6mL Tris stock for 100mM solution
 
#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>
##500mM*6mL=1000mM*x8##3mL
##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
##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
 
*'''[[User:Abigail E. Miller|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.
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:34, 7 October 2012 (EDT)''':why is this important? did this change your procedure?
 
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Revision as of 21:44, 6 December 2012

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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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