User:Matthew R Skorski/Notebook/471 - Exp BioChem/2015/11/04: Difference between revisions
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##The supernatant was removed but the fibers were retained | ##The supernatant was removed but the fibers were retained | ||
##Protinase K tube 11 was mixed with 1 mL of 1 mL of 50 mM pH 8 phosphate buffer | ##Protinase K tube 11 was mixed with 1 mL of 1 mL of 50 mM pH 8 phosphate buffer | ||
###Protinase K concentration: ( | ###Protinase K concentration: (0.00135 g)*(1mol/28,900g)*(1/0.001L)= 0.0000467 M Protinase K | ||
###Amount of Proteinase K solution needed for 1mL with | ###Proteinase K dilution: (46713 nM)*(0.1 mL Proteinase K) = (x nM)*(10 mL total dilution) => x = 467 nM | ||
###Amount of Buffer solution need to get to 1mL: (1mL total)-(0. | ###Proteinase K dilution: 0.1 mL of proteinase k solution was mixed with 9.9 mL of buffer | ||
###Amount of Proteinase K solution needed for 1mL with 10 nM concentration: M1*V1 = M2*V2 => (467 nM)*(V1) = (10 nM)*(1 mL) => V1 = 0.0214 mL | |||
###Amount of Buffer solution need to get to 1mL: (1mL total)-(0.0214 mL Protinase K solution) = 0.979 mL buffer | |||
#Incubating Samples | #Incubating Samples | ||
## | ##21.4 μL Protinase K and 979 μL buffer were mixed in the fiber tubes as well as blank eppendorf tubes | ||
##Eppendorf tubes were incubated on a shaker in a 37°C water bath for 25 minutes, 45 minutes, 75 minutes, 45 minutes, 105 minutes, 135 minutes, and 1440 minutes for one of the fiber tubes and one of the blanks. | ##Eppendorf tubes were incubated on a shaker in a 37°C water bath for 25 minutes, 45 minutes, 75 minutes, 45 minutes, 105 minutes, 135 minutes, and 1440 minutes for one of the fiber tubes and one of the blanks. | ||
#Running Samples | #Running Samples | ||
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###Excitation at 390 nm | ###Excitation at 390 nm | ||
###Emission from 400 to 650 nm | ###Emission from 400 to 650 nm | ||
###Both slit widths set to 10 nm | |||
###Scan Speed of 200 | |||
==Results== | ==Results== | ||
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[[Image:2015 11 4 Fluorescence Concentrations.png]] | [[Image:2015 11 4 Fluorescence Concentrations.png]] | ||
The 135 minute sample should be re-run as it is far too high of a value relative to the others. Also, more data points should be taken between 2 hours and 24 hours. Otherwise there is a general trend in increasing peptide concentration over time. | |||
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__NOTOC__ | __NOTOC__ |
Revision as of 20:11, 24 November 2015
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ObjectiveTo analyze the activity of proteinase k at 10 nM using fluorescence DescriptionThe guide for analyzing a protease with fluorescence can be found here at Dr. Hartings notebook
ResultsThe image below shows the intensity for the fluorescence of samples and blanks from 400 to 650 nm.
The 135 minute sample should be re-run as it is far too high of a value relative to the others. Also, more data points should be taken between 2 hours and 24 hours. Otherwise there is a general trend in increasing peptide concentration over time. |