Difference between revisions of "User:Melissa Novy/Notebook/CHEM-571/2012/10/24"

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(Assay Solution Concentrations and Volumes)
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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> solutions made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/10/23|2012/10/23]] were used.
 
** 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.
 
** 4-Iodophenol made on [[User:Melissa_Novy/Notebook/CHEM-571/2012/09/18|2012/09/18]] was 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==

Revision as of 15:24, 23 November 2012

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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] '
Trial Luminol H2O2 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
2 454 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
3 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 9.6 µM 2000 µL x
4 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 66 µL at 0.96 µM 2000 µL x
5 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 33 µL at 2.3 µM x 1000 µL
6 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 1000 µL
7 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 0.96 µM x 1000 µL
8 454 µL at 0.564 mM 112.7 µL at 5 mM 13.2 µL at 18 mM 16.5 µL at 2.3 µM x 2000 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] ' ' ' '
Trial Total Volume of Solution [µL] Luminol H2O2 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
8 2596.4 98.61962718 217.0312741 91.51132337 0.014616392 641.6576799

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.