Team:Toronto/Protocols

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<h1>'''Protocols'''</h1>
<h1>'''Protocols'''</h1>
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<h2>'''Tolerance Assay'''</h2>
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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).

Revision as of 21:52, 25 October 2010

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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).