User:Melissa Novy/Notebook/CHEM-571/2012/10/24

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==Objectives==
==Objectives==
 +
* Transform <i>E. coli</i> cells with K110A plasmids prepared on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/17|2012/10/17]].
* Optimize the HRP-luminol chemiluminescence assay.
* Optimize the HRP-luminol chemiluminescence assay.
-
** Carbonate buffer, luminol, HRP, and H<sub>2</sub>O<sub>2</sub>  
+
** Carbonate buffer, luminol, HRP, and H<sub>2</sub>O<sub>2</sub> solutions made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/23|2012/10/23]] were used.
-
solutions made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/23|2012/10/23]] were used.
+
** 4-Iodophenol made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18|2012/09/18]] was used.
 +
 
 +
==Cell Transformation==
 +
* There were no deviations from the recommended [[AU_Biomaterials_Design_Lab:Protocols/Transformation_Protocol|transformation protocol]], with the exception of the following:
 +
* 200 μL of SOC media was added to one solution of cells, while 200 μL LB media was added to the other solution of cells before being shaken at 275 rpm for 1 hr.
 +
* 25 μL of transformed cells was plated on one Petri dish, while 200 μL of transformed cells was plated on the other.
 +
** The plates were pre-warmed and contained LB and kanamycin.
 +
** Two plates contained cells grown in SOC media, while the other two plates contained cells grown in LB media.
==Assay Solution Concentrations and Volumes==
==Assay Solution Concentrations and Volumes==
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| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
|-
-
| Trial||Luminol||H2O2||4-Iodophenol||HRP||Water||Carbonate Buffer
+
| Trial||Luminol||H<sub>2</sub>O<sub>2</sub>||4-Iodophenol||HRP||Water||Carbonate Buffer
|-
|-
| 1||454 µL at 0.564 mM||112.7 µL at 5 mM||13.2 µL at 18 mM||33 µL at 9.6 µM||1033 µL||x
| 1||454 µL at 0.564 mM||112.7 µL at 5 mM||13.2 µL at 18 mM||33 µL at 9.6 µM||1033 µL||x
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|}
|}
-
* Note that the concentration of the carbonate buffer was ? M and pH 8.5.
+
* Note that the concentration of the carbonate buffer was 0.833 M and pH 8.5.
* Trials 5 through 8 used another HRP solution that was diluted with carbonate buffer instead of deionized water.
* Trials 5 through 8 used another HRP solution that was diluted with carbonate buffer instead of deionized water.
** 25 μL of 9.6 μM HRP in water was added to 75 μL of carbonate buffer to produce a solution of 2.3 μM HRP in carbonate buffer.
** 25 μL of 9.6 μM HRP in water was added to 75 μL of carbonate buffer to produce a solution of 2.3 μM HRP in carbonate buffer.
** 417.4 μL of this 2.3 μM HRP solution in carbonate buffer was then added to 582.6 μL of carbonate buffer to produce a solution of 0.96 μM HRP in carbonate buffer.
** 417.4 μL of this 2.3 μM HRP solution in carbonate buffer was then added to 582.6 μL of carbonate buffer to produce a solution of 0.96 μM HRP in carbonate buffer.
* Trials 1 through 4 were diluted with water, while trials 5 through 8 were diluted with carbonate buffer.
* Trials 1 through 4 were diluted with water, while trials 5 through 8 were diluted with carbonate buffer.
 +
 +
{| {{table}}
 +
| align="center" style="background:#f0f0f0;"|''''''
 +
| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|'''Final Concentration in Cuvette [µM]'''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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| align="center" style="background:#f0f0f0;"|''''''
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|-
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| Trial||Total Volume of Solution [µL]||Luminol||H<sub>2</sub>O<sub>2</sub>||4-Iodophenol||HRP||Carbonate Buffer
 +
|-
 +
| 1||1645.9||155.5720275||342.3658789||144.3587095||0.192478279||x
 +
|-
 +
| 2||1645.9||155.5720275||342.3658789||144.3587095||0.192478279||x
 +
|-
 +
| 3||2612.9||97.99686172||215.6607601||90.9334456||0.121244594||x
 +
|-
 +
| 4||2645.9||96.77463245||212.9710118||89.79931214||0.023946483||x
 +
|-
 +
| 5||1612.9||158.7550375||349.3706987||147.3122946||0.047058094||516.4610329
 +
|-
 +
| 6||1596.4||160.3958908||352.9817088||148.8348785||0.023772238||521.7990479
 +
|-
 +
| 7||1596.4||160.3958908||352.9817088||148.8348785||0.009922325||521.7990479
 +
|-
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| 8||2596.4||98.61962718||217.0312741||91.51132337||0.014616392||641.6576799
 +
|}
==Emission Spectra and Data Analysis==
==Emission Spectra and Data Analysis==

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Objectives

  • Transform E. coli cells with K110A plasmids prepared on 2012/10/17.
  • Optimize the HRP-luminol chemiluminescence assay.
    • Carbonate buffer, luminol, HRP, and H2O2 solutions made on 2012/10/23 were used.
    • 4-Iodophenol made on 2012/09/18 was used.

Cell Transformation

  • There were no deviations from the recommended transformation protocol, with the exception of the following:
  • 200 μL of SOC media was added to one solution of cells, while 200 μL LB media was added to the other solution of cells before being shaken at 275 rpm for 1 hr.
  • 25 μL of transformed cells was plated on one Petri dish, while 200 μL of transformed cells was plated on the other.
    • The plates were pre-warmed and contained LB and kanamycin.
    • Two plates contained cells grown in SOC media, while the other two plates contained cells grown in LB media.

Assay Solution Concentrations and Volumes

' Volume and Stock Concentration Added to Cuvette ' ' ' Volume Added to Cuvette [µL] '
TrialLuminolH2O24-IodophenolHRPWaterCarbonate Buffer
1454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM1033 µLx
2454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM1033 µLx
3454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 9.6 µM2000 µLx
4454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM66 µL at 0.96 µM2000 µLx
5454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM33 µL at 2.3 µMx1000 µL
6454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 2.3 µMx1000 µL
7454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 0.96 µMx1000 µL
8454 µL at 0.564 mM112.7 µL at 5 mM13.2 µL at 18 mM16.5 µL at 2.3 µMx2000 µL
  • Note that the concentration of the carbonate buffer was 0.833 M and pH 8.5.
  • Trials 5 through 8 used another HRP solution that was diluted with carbonate buffer instead of deionized water.
    • 25 μL of 9.6 μM HRP in water was added to 75 μL of carbonate buffer to produce a solution of 2.3 μM HRP in carbonate buffer.
    • 417.4 μL of this 2.3 μM HRP solution in carbonate buffer was then added to 582.6 μL of carbonate buffer to produce a solution of 0.96 μM HRP in carbonate buffer.
  • Trials 1 through 4 were diluted with water, while trials 5 through 8 were diluted with carbonate buffer.
' ' Final Concentration in Cuvette [µM] ' ' ' '
TrialTotal Volume of Solution [µL]LuminolH2O24-IodophenolHRPCarbonate Buffer
11645.9155.5720275342.3658789144.35870950.192478279x
21645.9155.5720275342.3658789144.35870950.192478279x
32612.997.99686172215.660760190.93344560.121244594x
42645.996.77463245212.971011889.799312140.023946483x
51612.9158.7550375349.3706987147.31229460.047058094516.4610329
61596.4160.3958908352.9817088148.83487850.023772238521.7990479
71596.4160.3958908352.9817088148.83487850.009922325521.7990479
82596.498.61962718217.031274191.511323370.014616392641.6576799

Emission Spectra and Data Analysis

  • Please refer to Dhea Patel's entry for an emission spectrum taken at 425 nm of trial 6 of the chemiluminescence assay.
  • Note that all trials were conducted over a time period of 200 s in a darkened room. The cuvette containing all of the components of the reaction solution, with the exception of the 5 mM H2O2 solution, were placed in the sample cell. The detector was activated, and then the 5 mM H2O2 solution was pipetted into the cuvette to begin the reaction.
  • The time difference between the activation of the detector and the start of the reaction can be seen in the spectrum of trial 6. It appears that luminescence was first registered by the instrument at about 8 seconds. However, the intensity quickly decreased over a period of about 7 seconds and returned to a near-baseline intensity.
  • The reason for using the spectrum of trial 6 is that it produced the longest-lasting luminescence of all trials. Therefore, it must be assumed that diluting assay solutions with carbonate buffer and using HRP diluted in carbonate buffer at a relatively lower concentration than before will produce more useful results.
  • If further tests are conducted, it may be advantageous to dilute the assay solutions with carbonate buffer at higher and lower concentrations.




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