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

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Starter cultures were centrifuged for 15 minutes at 4500rpm and resuspended in 4mL LB broth.
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.
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''': more than that was done. those cells were then added to the 1L fernbach flasks. what time did this all occur? you need to link ot Mary's notebook since she is the one who actually did the experimental procedure.
## 1mL .4M IPTG was added to each flask to start protein exprssion
## 1mL .4M IPTG was added to each flask to start protein exprssion
## Expression cultures were incubated at 160rpm and 37°C for 4 hours.
## Expression cultures were incubated at 160rpm and 37°C for 4 hours.
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':be more precise. at exactly what time was hte IPTG added? what time were hte expression cultures removed?
## 30mL binding buffer was inserted to each flask.
## 30mL binding buffer was inserted to each flask.
## Flasks were centrifigued at 4500rpm for 15 minutes
## Flasks were centrifigued at 4500rpm for 15 minutes
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':binding buffer  was used to resuspend cells after centrifugation. you need to link to someone's notebook if you did not do it.
## Cells were collected and placed in the freezer at -80°C.
## Cells were collected and placed in the freezer at -80°C.
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':you need more details.


#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
*'''[[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
##.02M Tris solution *1L = .02moles Tris needed
##.02mol Tris * 121.14g = 2.4228g Tris weighed
##.02mol Tris * 121.14g = 2.4228g Tris weighed
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#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
*'''[[User:Abigail E. Miller|Abigail E. Miller]] 11:32, 7 October 2012 (EDT)''':see above comment
##.02M Tris solution *.5L = .01moles Tris needed
##.02M Tris solution *.5L = .01moles Tris needed
##.01mol Tris * 121.14g = 1.2114g Tris
##.01mol Tris * 121.14g = 1.2114g Tris
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#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
#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 08:32, 7 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. Procedure for protein expression was followed with noted changes, and can be found here: AU_Biomaterials_Design_Lab:Protocols/Protein_Expression
    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.
  • Abigail E. Miller 11:32, 7 October 2012 (EDT): more than that was done. those cells were then added to the 1L fernbach flasks. what time did this all occur? you need to link ot Mary's notebook since she is the one who actually did the experimental procedure.
    1. 1mL .4M IPTG was added to each flask to start protein exprssion
    2. Expression cultures were incubated at 160rpm and 37°C for 4 hours.
  • Abigail E. Miller 11:32, 7 October 2012 (EDT):be more precise. at exactly what time was hte IPTG added? what time were hte expression cultures removed?
    1. 30mL binding buffer was inserted to each flask.
    2. Flasks were centrifigued at 4500rpm for 15 minutes
  • Abigail E. Miller 11:32, 7 October 2012 (EDT):binding buffer was used to resuspend cells after centrifugation. you need to link to someone's notebook if you did not do it.
    1. 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
  • Abigail E. Miller 11:32, 7 October 2012 (EDT):no made with...those are the exact chemicals and concentrations of the buffer.
    1. .02M Tris solution *1L = .02moles Tris needed
    2. .02mol Tris * 121.14g = 2.4228g Tris weighed
    3. .5M NaCl *1L =.5mol NaCl needed
    4. .5mol*58.439=29.2195g NaCl
    5. 29.2147gNaCl weighed
    6. 30mM imidazole *1L = .03mol imidazole needed
    7. .03mol*68.077g/mol=2.0423g imidazole weighed
    8. 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
  • 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.
    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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