Cconboy:Terminator Characterization/Methods: Difference between revisions
No edit summary |
No edit summary |
||
| Line 2: | Line 2: | ||
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. | 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). | Flow cytometry was performed using a FACS Aria machine 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=== | ||
Latest revision as of 13:03, 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 machine 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)
