User:Andrew W Long/Notebook/CHEM-671 Research/2015/11/11
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1 nM Fluorescence Assay
The detailed instructions for today’s procedure can be found here. First 7 AuNP fiber samples were prepped by spinning them at 300rpm for 10min. The supernatant was then removed. Next 1mL of Tris/CaCl buffer was added to proteinase-K stock 11, making the concentration XXM proteinase-K. The amount of proteinase-K solution required to make the final reaction volume 1mL with a 1uM starting concentration of proteinase-K was determined to be XXmL. The amount of buffer needed to bring the total reaction volume to 1mL was determined to be (1mL total) - (XXmL proteinase-K solution) = XXmL Tris-CaCl. XXuL proteinase-K and XXuL buffer were then added to each of the 7 drained fiber samples, as well as 7 blanks (to be subtracted later in the data correction phase). The resulting 14 solutions were then incubated in a hot water shaker for each of their respective times (10min, 15min, 30min, 45min, 60min, 90min, and 120min). When removed from the bath, each sample is centrifuged for 12000rpm for 1min to collect any undigested fibers. 20uL of the reaction mixture was taken and mixed with 140uL fluorometric assay buffer, and 40uL fluorometric assay reagent in a 600uL eppendorf tube. The resulting solution was then examined using an excitation wavelength of 390nm, and scanned for emission from 400-650nm. The results of the assay are shown below. The calibration curve used to determine the peptide concentration in the final solution can be found 10/07/15|here.