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

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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.

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

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.

@@ 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.
 ** 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==