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Team:Toronto/Protocols
From 2010.igem.org
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Protocols
Tolerance Assay
A 0.5 M (10x) stock solution of catechol was made in a 5 ml volume of autoclaved, distilled water. This solution was added to 25 ml of sterile MM2 media to make up a volume of 30 ml. This was added to 20 ml of MM2 innoculated with E.coli DH5a (approximate density 1x10e8 cells per ml) for a final volume of 50 ml (catechol concentration 50mM). Control samples were created as above minus catechol. Treated and untreated samples were placed in a 37C shaker incubator and aliquots were removed every two hours. 0.5 ml aliquots were serially diluted in 4.5 ml of MM2 media and four dilutions representing 1 x 10-3, 1 x 10-4, 1 x 10-5 and 1x10-6 were plated by spreading 200 ul of the diluted sample on LB agar plates. After drying, plates were incubated in an inverted position in a 37C incubator overnight. Plates with between 30 and 300 colonies were counted to determine the number of colony forming units using the method described by Imperial College (iGEM 2009 OpenWetWare).
Making Chemically Competent Cells
Day 1
1. Start a 5 mL LB overnight culture in the 37C incubator from either a colony streaked plate or with one of the last frozen competent stocks of the strains you want to make competent.
Day 2
1. Inoculate the 5 mL overnight culture into a new flask with 200 mL of fresh LB.
2. Grow until O.D. 600 = 0.4.
a. To check O.D., take 1 mL of the bacterial culture and put it in a plastic cuvette.
b. Take another plastic cuvette and add in 1 mL of LB.
c. Change the wavelength of the spectrophotometer to 600 nm.
d. Blank using the LB cuvette.
e. Measure and record the absorbance of the cuvette containing the culture.
f. If the O.D. is below 0.4, place the flask back in the 37°C incubator and repeat ‘e’ at a later time point. Dispose of the cuvette with the culture but keep the cuvette with the LB as a blank.
3. Transfer 50 mL of the culture into a pre-chilled Falcon tube. Repeat for each of the remaining 3 Falcon tubes.
4. Place the Falcon tubes in ice for 15 minutes to cool the cultures.
5. Pellet the cells using a Beckman centrifuge at 4000 rpm for 10 minutes at 4°C.
