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

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==Objectives==
*begin protein expression
*make elution and binding buffers
*collect E. Coli with ADA
==Entry title==
==Entry title==
#Expressing ADA
##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.
## 1mL .4M IPTG was added to each flask to start protein exprssion
## Expression cultures were incubated at 160rpm and 37°C for 4 hours.
## 30mL binding buffer was inserted to each flask.
## Flasks were centrifigued at 4500rpm for 15 minutes
## Cells were collected and placed in the freezer at -80°C.
#Elution Buffer
#Elution Buffer
#.02M Tris solution was needed for 10mL buffer.
#.02M Tris solution was needed for 10mL buffer.

Revision as of 11:44, 1 October 2012

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Objectives

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

Entry title

  1. Expressing ADA
    1. Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
    2. To inoculate the expression culture, resuspended cells were divided between flasks.
    3. 1mL .4M IPTG was added to each flask to start protein exprssion
    4. Expression cultures were incubated at 160rpm and 37°C for 4 hours.
    5. 30mL binding buffer was inserted to each flask.
    6. Flasks were centrifigued at 4500rpm for 15 minutes
    7. Cells were collected and placed in the freezer at -80°C.
  1. Elution Buffer
  2. .02M Tris solution was needed for 10mL buffer.
    1. .02M Tris * .01L = .0002moles/liter
    2. .0002mol *121.14= .02423g Tris
    3. .249 was the actual amount weighed and put in 7mL water.
    4. HCl and NaOH were added dropwise until the pH was 7.53, between a target range of 7-8.
  3. .4g IPTG was weighed and placed in 4mL of buffer

1mL of IPTG solution was added to each expression culture.

  1. Buffer for binding was made with 20mM Tris, .5M NaOH and 20-40mM amidazole at a pH of 7.4
    1. .02M Tris solution *1L = .02moles Tris
    2. .02mol Tris * 121.14g = 2.4228g Tris
    3. .5M NaCl *1L =.5mol
    4. .5mol*58.439=29.2195gNaCl
    5. 29.2147gNaCl weighed
    6. 30mM imidazole *1L = .03mol imidazole
    7. .03mol*68.077g/mol=2.0423g imidazole
    8. 2.4721g Tris 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
    2. .01mol Tris * 121.14g = 1.2114g Tris
    3. 1.2164g Tris weighed
    4. .5M NaCl *.5 = .25mol
    5. .25mol*58.439g/mol=14.6098gNaCl
    6. .5M imidazole *.5L = .25mol imidazole
    7. .25mol*68.077g/mol=17.019g imidazole
  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
    5. 200mM*6mL=1000mM*x
    6. 1.2mL
    7. 100mM*6mL=1000mM*x
    8. .6mL
    9. 50mM*6mL=1000mM*x
    10. .3mL
    11. 5mM*6mL=1000mM*x
    12. .03mL
    13. .5mM*6mL=1000mM*x
    14. .003mL
    15. .05mM*6mL=1000mM*x
    16. .0003mL



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