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

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==Procedure==
==Procedure==
#Expressing ADA
#Expressing ADA
##Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
## AU_Biomaterials_Design_Lab:Protocols/Protein_Expression procedure was followed.
## Tris stock for 1M solution
Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
## To inoculate the expression culture, resuspended cells were divided between flasks.
## To inoculate the expression culture, resuspended cells were divided between flasks.
## 1mL .4M IPTG was added to each flask to start protein exprssion
## 1mL .4M IPTG was added to each flask to start protein exprssion
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#Binding buffer  
#Binding buffer  
##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
##made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
##.02M Tris solution *1L = .02moles Tris
##.02M Tris solution *1L = .02moles Tris needed
##.02mol Tris * 121.14g = 2.4228g Tris
##.02mol Tris * 121.14g = 2.4228g Tris weighed
##.5M NaCl *1L =.5mol
##.5M NaCl *1L =.5mol NaCl needed
##.5mol*58.439=29.2195gNaCl
##.5mol*58.439=29.2195g NaCl
##29.2147gNaCl weighed
##29.2147gNaCl weighed
##30mM imidazole *1L = .03mol imidazole
##30mM imidazole *1L = .03mol imidazole needed
##.03mol*68.077g/mol=2.0423g imidazole
##.03mol*68.077g/mol=2.0423g imidazole weighed
##2.4721g Tris weighed
##HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
##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
#A buffer for elution was made with 20mM Tris, .5M NaCl and 500mM amidazole
##.02M Tris solution *.5L = .01moles Tris
##.02M Tris solution *.5L = .01moles Tris needed
##.01mol Tris * 121.14g = 1.2114g Tris
##.01mol Tris * 121.14g = 1.2114g Tris
##1.2164g Tris weighed
##1.2164g Tris weighed
##.5M NaCl *.5 = .25mol
##.5M NaCl *.5 = .25mol NaCl needed
##.25mol*58.439g/mol=14.6098gNaCl
##.25mol*58.439g/mol=14.6098gNaCl weighed
##.5M imidazole *.5L = .25mol imidazole
##.5M imidazole *.5L = .25mol imidazole needed
##.25mol*68.077g/mol=17.019g imidazole
##.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.
#HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.


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##500mM*6mL=1000mM*x8##3mL
##500mM*6mL=1000mM*x8##3mL
##1000mM*6mL=1000mM*x
##1000mM*6mL=1000mM*x
##6mL  
##6mL Tris stock for 1M solution
##200mM*6mL=1000mM*x
##200mM*6mL=1000mM*x
##1.2mL
##1.2mL Tris stock for 200mM solution
##100mM*6mL=1000mM*x
##100mM*6mL=1000mM*x
##.6mL
##.6mL Tris stock for 100mM solution
##50mM*6mL=1000mM*x
##50mM*6mL=1000mM*x
##.3mL
##.3mL Tris stock for 50mM solution
##5mM*6mL=1000mM*x
##5mM*6mL=1000mM*x
##.03mL
##.03mL Tris stock for 5mM solution
##.5mM*6mL=1000mM*x
##.5mM*6mL=1000mM*x
##.003mL
##.003mL Tris stock for .5mM solution
##.05mM*6mL=1000mM*x
##.05mM*6mL=1000mM*x
##.0003mL
##.0003mL Tris stock for .05mM solution


==Observations==
==Observations==

Revision as of 21:17, 4 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. AU_Biomaterials_Design_Lab:Protocols/Protein_Expression procedure was followed.
    2. Tris stock for 1M solution

Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.

    1. To inoculate the expression culture, resuspended cells were divided between flasks.
    2. 1mL .4M IPTG was added to each flask to start protein exprssion
    3. Expression cultures were incubated at 160rpm and 37°C for 4 hours.
    4. 30mL binding buffer was inserted to each flask.
    5. Flasks were centrifigued at 4500rpm for 15 minutes
    6. Cells were collected and placed in the freezer at -80°C.
  1. Binding buffer
    1. made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
    2. .02M Tris solution *1L = .02moles Tris needed
    3. .02mol Tris * 121.14g = 2.4228g Tris weighed
    4. .5M NaCl *1L =.5mol NaCl needed
    5. .5mol*58.439=29.2195g NaCl
    6. 29.2147gNaCl weighed
    7. 30mM imidazole *1L = .03mol imidazole needed
    8. .03mol*68.077g/mol=2.0423g imidazole weighed
    9. HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.
  1. A buffer for elution was made with 20mM Tris, .5M NaCl and 500mM amidazole
    1. .02M Tris solution *.5L = .01moles Tris needed
    2. .01mol Tris * 121.14g = 1.2114g Tris
    3. 1.2164g Tris weighed
    4. .5M NaCl *.5 = .25mol NaCl needed
    5. .25mol*58.439g/mol=14.6098gNaCl weighed
    6. .5M imidazole *.5L = .25mol imidazole needed
    7. .25mol*68.077g/mol=17.019g imidazole weighed
  2. HCl and NaOH were added dropwise until the pH was 7.4 as indicated by a pH meter.


  1. Tris buffer stock solution was inserted into HAuCl4 and BSA tubes at molarities of .05mM, .5mM, 5mM, 50mM, 100mM, 200mM, 500mM and 1M
    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

Observations

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


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