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Life is a great big canvas, and you should
throw all the paint on it you can.
Danny Kaye
Day 6: Functional analysis of simple circuit;
transformation with two plasmids
Assignments Due
Overview of Experiment
Today you measure fluorescence output of pTetR-GFP (transformation
with BioBrick ligation) and transform bacteria with two plasmids.
Functional analysis of simple circuit (pTetR-GFP)
Experimental goal: Does the genetic circuit work?
- Outline of goals: How do you get a big signal?
- What are appropriate controls?
- How do we account for background?
- How does signal noise affect the results?
- Fluorescence signal detection
- pellet, wash, and resuspend cells
- determine density of cultures
- determine fluorescence output
Bacterial transformation with two plasmids
- Plasmid DNA
- pET-Venus-GRX2 (Kan resistant)
- pTara (chloramphenicol (Chl) resistant)
- NEB 5-alpha Competent E. coli (Subcloning Efficiency)
- perform ONE double transformation per TEAM (follow the manufacturer's protocol)
- use 1 µl of each plasmid
- spread using glass beads on LB-Kan (10 µg/ml)/ Chl (10 µg/ml)
supplemented with 0.2% glucose: pipet 50 µl transformation
and 50 µl
SOC and spread on 1st plate; spread 100 µl
transformation on a 2nd plate
Confirmation of Isothermal Assembly Transformation
YOU WILL HAVE TO COME IN THE DAY BEFORE LAB DAY 7 AND
PULL FOUR COLONIES (assembled vector) per team.
The colonies will be grown overnight in 3 ml LB-Amp (50 µg/ml)/Kan (20 µg/ml) at 37°C with shaking (225 rpm).
Copyright, Acknowledgements,
and Intended Use
Created by B. Beason (bbeason@rice.edu),
Rice University, 21 November 2007
Updated 2 November 2011