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

Melissa Novy: /* Cell Transformation */

2012-11-23T22:24:44Z

Cell Transformation

←Older revision		Revision as of 22:24, 23 November 2012
Line 26:		Line 26:
	* 25 μL of transformed cells was plated on one Petri dish, while 200 μL of transformed cells was plated on the other.		* 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.		** 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==

Melissa Novy: /* Cell Transformation */

2012-10-26T05:53:29Z

Cell Transformation

←Older revision		Revision as of 05:53, 26 October 2012
Line 22:		Line 22:

	==Cell Transformation==		==Cell Transformation==
-	* There were no deviations from the recommended[[AU_Biomaterials_Design_Lab:Protocols/Transformation_Protocol\|transformation protocol]], with the exception of the following:	+	* There were no deviations from the recommended [[AU_Biomaterials_Design_Lab:Protocols/Transformation_Protocol\|transformation protocol]], with the exception of the following:
-	* SOC media was added to one solution of cells, while LB media was added to the other ~~tube~~.	+	* 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 ~~250~~ μL of transformed cells was plated on the other.	+	* 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.

	==Assay Solution Concentrations and Volumes==		==Assay Solution Concentrations and Volumes==

Melissa Novy at 05:51, 26 October 2012

2012-10-26T05:51:28Z

←Older revision		Revision as of 05:51, 26 October 2012
Line 16:		Line 16:

	==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:
		+	* SOC media was added to one solution of cells, while LB media was added to the other tube.
		+	* 25 μL of transformed cells was plated on one Petri dish, while 250 μL of transformed cells was plated on the other.

	==Assay Solution Concentrations and Volumes==		==Assay Solution Concentrations and Volumes==

Melissa Novy: /* Assay Solution Concentrations and Volumes */

2012-10-26T05:33:09Z

Assay Solution Concentrations and Volumes

←Older revision		Revision as of 05:33, 26 October 2012
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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;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|'''Final Concentration in Cuvette [µM]'''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\|-
		+	\| 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
		+	\|-
		+	\| 8\|\|2596.4\|\|98.61962718\|\|217.0312741\|\|91.51132337\|\|0.014616392\|\|641.6576799
		+	\|}

	==Emission Spectra and Data Analysis==		==Emission Spectra and Data Analysis==

Melissa Novy: /* Objectives */

2012-10-26T04:50:16Z

Objectives

←Older revision		Revision as of 04:50, 26 October 2012
Line 18:		Line 18:
	* 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.

	==Assay Solution Concentrations and Volumes==		==Assay Solution Concentrations and Volumes==

Melissa Novy: /* Objectives */

2012-10-26T04:47:22Z

Objectives

←Older revision		Revision as of 04:47, 26 October 2012
Line 17:		Line 17:
	==Objectives==		==Objectives==
	* 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.	+

	==Assay Solution Concentrations and Volumes==		==Assay Solution Concentrations and Volumes==

Melissa Novy at 04:47, 26 October 2012

2012-10-26T04:47:03Z

←Older revision		Revision as of 04:47, 26 October 2012
Line 15:		Line 15:
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->

-	==~~Entry title~~==	+	==Objectives==
-	* ~~Insert content here~~...	+	* 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.
		+
		+	==Assay Solution Concentrations and Volumes==
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume and Stock Concentration Added to Cuvette'''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume Added to Cuvette [µL]'''
		+	\| align="center" style="background:#f0f0f0;"\|''''''
		+	\|-
		+	\| 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 ? 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.
		+
		+	==Emission Spectra and Data Analysis==
		+	* Please refer to [[User:Dhea_Patel/Notebook/Experimental_Biological_Chemistry_Notebook/2012/10/24\|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 H<sub>2</sub>O<sub>2</sub> solution, were placed in the sample cell. The detector was activated, and then the 5 mM H<sub>2</sub>O<sub>2</sub> 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.
		+


	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
-	\|}	+

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Melissa Novy: Autocreate 2012/10/24 Entry for User:Melissa_Novy/Notebook/CHEM-571

2012-10-26T04:11:58Z

Autocreate 2012/10/24 Entry for User:Melissa_Novy/Notebook/CHEM-571

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==Entry title==
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