Cconboy:Terminator Characterization/Methods: Difference between revisions
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===Sample Preparation=== | ===Sample Preparation=== | ||
Overnights were inoculated from plated colonies in 5mL of LB media with | Overnights were inoculated from plated colonies in 5mL of LB media with 50?g/mL Ampicillin. Samples were grown at 37C. In late log phase (OD600~1.5-2) 1mL of culture was pelleted, resuspended in 1mL 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 405nm for CFP and 480nm for YFP. CFP fluorescence was detected using a 475/85 filter (i.e. 475 +/-43nm). YFP fluorescence was detected with a 530/30 filter (i.e. 530 +/-30nm). | Flow cytometry was performed using a FACS Aria equipped with two lasers, providing excitation at 405nm for CFP and 480nm for YFP. CFP fluorescence was detected using a 475/85 filter (i.e. 475 +/-43nm). YFP fluorescence was detected with a 530/30 filter (i.e. 530 +/-30nm). | ||
===Analysis=== | ===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 PoPSin/PoPSout. Termination efficiency is consequently defined as one minus the terminator read-through: | |||
(1) Termination efficiency = 1 � (mean YFP/mean CFP*2.96) | |||
'''Single Cell Termination Efficiency:''' | |||
See Jason Kelly�s eventual documentation of this method. | |||
Revision as of 14:51, 23 March 2006
Page in Progress
Sample Preparation
Overnights were inoculated from plated colonies in 5mL of LB media with 50?g/mL Ampicillin. Samples were grown at 37C. In late log phase (OD600~1.5-2) 1mL of culture was pelleted, resuspended in 1mL 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 405nm for CFP and 480nm for YFP. CFP fluorescence was detected using a 475/85 filter (i.e. 475 +/-43nm). 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 PoPSin/PoPSout. Termination efficiency is consequently defined as one minus the terminator read-through:
(1) Termination efficiency = 1 � (mean YFP/mean CFP*2.96)
Single Cell Termination Efficiency: See Jason Kelly�s eventual documentation of this method.
