User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/10/24

Experimental Biological Chemistry I

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Purpose To test luminol assay by varying the concentrations of H2O2, Luminol, and horseradish peroxidase DH5α-T1 E.Coli bacteria cells were transformed with E34K plasmids, and transformed bacteria were plated on LB with kanamycin for cell growth Procedure Transformation DH5α-T1 E.Coli bacteria were defrosted after taken out from freezer and defrosted by placing on ice. ADA plasmid with E34K mutation was taken our of freezer and defrost by placing on ice. 40uL of bacteria were placed in a 1.5mL microcentrifuge tube and mixed with 5uL of ADA E34K plasmid. Two tubes of the culture was placed on heat block at 42°C for 30 seconds, and immediately placed back on ice. 200uL of pre-warmed SOC was added to one of 45uL culture. 200uL of sterile LB was added to the other 45uL culture. Both microcentrifuge tube including the 250uL culture was taped to test tubes and placed on orbital shaker for 37°C for 1 hour. After 1 hour in shaker, cells were taken out. For each microcentrifuge tube, 200uL of culture was plated onto a pre-warmed LB and kanamycin plate, and the left-over 50uL of culture was plated onto another pre-warmed LB and kanamycin plate. Four plates were plated as a result: two plates plated with cells grew in SOC medium, and other two plates were plated with cells grew in LB medium. The plates were placed in incubator at 37°C for overnight, and taken out the next morning. Luminol Assay Different volumes and concentration of H2O2, luminol, and HRP were added for each samples Small portable fluorometer was used instead of fluorometer used in 09/26/2012 Samples were run in dark room to minimize the amount of background noise produced by environment The solutions used for making all samples were prepared on 10/23/2012 For details on each concentration and volumes of H2O2, luminol, and horseradish peroxidase were depicted in a table in Melissa's Notebook, which records the final concentration of all substrates and enzymes involved in each sample. Results Sample 5 to 8 were made with carbonate buffer as oppose to water. The usage of carbonate buffer increases the luminescence lifetime of the luminol assay. This suggests the use of carbonate buffer assists the reaction of luminol with H2O2. Horseradish peroxidase were diluted to different concentrations during sample testing. However, after comparison of each fluorescence peak, HRP that yields the greatest luminescence lifetime is 2.3uM. Because the data obtained from each trial were not kept, no results were shown for this sets of data.