Cconboy:Terminator Characterization/Methods: Difference between revisions
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===Sample Preparation=== | ===Sample Preparation=== | ||
Overnight cultures were inoculated from plated colonies in 5 mL of LB medium with 50 ug/mL Ampicillin. Samples were grown at 37 C. In late log phase (OD600 ~1.5-2) 1 mL of culture was pelleted, resuspended in 1 mL PBS, and stored on ice for analysis by flow cytometry. | |||
Flow cytometry was performed using a FACS Aria equipped with two lasers, providing excitation at | Flow cytometry was performed using a FACS Aria equipped with two lasers, providing excitation at 405 nm for CFP and 480 nm for YFP. CFP fluorescence was detected using a 475/85 filter (''i.e.'', 475 +/-43 nm). YFP fluorescence was detected with a 530/30 filter (''i.e.'', 530 +/-30nm). | ||
===Analysis=== | ===Analysis=== | ||
'''Population Average Termination Efficiency:''' | '''Population Average Termination Efficiency:''' | ||
Average terminator read-through was derived from the mean fluorescence ratio of YFP/CFP, normalized to the ratio of the no terminator control construct (BBa_I7003). This analysis makes the assumption that the average YFP/CFP ratio is correlated with the ratio of | Average terminator read-through was derived from the mean fluorescence ratio of YFP/CFP, normalized to the ratio of the no-terminator control construct (BBa_I7003). This analysis makes the assumption that the average YFP/CFP ratio is correlated with the ratio of PoPS-in/PoPS-out. Termination efficiency is consequently defined as one minus the terminator read-through: | ||
Termination efficiency = 1 - (mean YFP/mean CFP*2.96) | Termination efficiency = 1 - (mean YFP/mean CFP*2.96) | ||
Revision as of 13:02, 15 February 2007
Sample Preparation
Overnight cultures were inoculated from plated colonies in 5 mL of LB medium with 50 ug/mL Ampicillin. Samples were grown at 37 C. In late log phase (OD600 ~1.5-2) 1 mL of culture was pelleted, resuspended in 1 mL PBS, and stored on ice for analysis by flow cytometry.
Flow cytometry was performed using a FACS Aria equipped with two lasers, providing excitation at 405 nm for CFP and 480 nm for YFP. CFP fluorescence was detected using a 475/85 filter (i.e., 475 +/-43 nm). YFP fluorescence was detected with a 530/30 filter (i.e., 530 +/-30nm).
Analysis
Population Average Termination Efficiency: Average terminator read-through was derived from the mean fluorescence ratio of YFP/CFP, normalized to the ratio of the no-terminator control construct (BBa_I7003). This analysis makes the assumption that the average YFP/CFP ratio is correlated with the ratio of PoPS-in/PoPS-out. Termination efficiency is consequently defined as one minus the terminator read-through:
Termination efficiency = 1 - (mean YFP/mean CFP*2.96)
