User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/02/10: Difference between revisions
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== | ==Proteinase K Reaction Analysis== | ||
==Objectives== | |||
#In situ 100 nM Proteinase K reaction | |||
#In vitro reaction to generate samples for Bradford analysis and gel electrophoresis | |||
#Generate a Bradford calibration curve | |||
==Protocol== | |||
#'''In situ reaction:''' | |||
0.0075 mL Proteinase K stock was added to 2.9925 mL Tris/NaCl buffer for a final enzyme concentration of 100 nM. Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted; the fibers were resuspended and added to the cuvette. | |||
The Ocean Optics was set to scan every 30 seconds for 2 hours, and maintained at 37°C and stirred at 1000 RPM. | |||
#'''Proteinase K reaction for analysis''' | |||
[http://openwetware.org/wiki/User:Monika_Gasiorek/Notebook/CHEM-571_2014F/2015/02/10 Monika] has values for the samples prepped. Samples were run at 37°C for 2.5 hours. 300 µL of sample was extracted every 30 minutes | |||
#'''Bradford Calibration Curve''' | |||
[http://openwetware.org/wiki/User:Monika_Gasiorek/Notebook/CHEM-571_2014F/2015/02/10 Monika] has the data for the calibration curve samples. A 1 in 4 Bradford solution was prepared with Tris/NaCl buffer, and a calibration curve was generated. | |||
'''Results will be posted in tomorrow's page.''' | |||
Revision as of 15:44, 10 February 2015
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Proteinase K Reaction AnalysisObjectives
Protocol
0.0075 mL Proteinase K stock was added to 2.9925 mL Tris/NaCl buffer for a final enzyme concentration of 100 nM. Fibers were spun down at 300 RPM for 5 minutes, and liquid extracted; the fibers were resuspended and added to the cuvette. The Ocean Optics was set to scan every 30 seconds for 2 hours, and maintained at 37°C and stirred at 1000 RPM.
Monika has values for the samples prepped. Samples were run at 37°C for 2.5 hours. 300 µL of sample was extracted every 30 minutes
Monika has the data for the calibration curve samples. A 1 in 4 Bradford solution was prepared with Tris/NaCl buffer, and a calibration curve was generated. Results will be posted in tomorrow's page.
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