Competence Assays

Day 0: Inoculation

• Prepare n+1 tubes with 5ml LB
• Inoculate tubes with either 2ul of liquid culture or just get pipette tip with frozen culture in there.
• Incubate overnight at 30° C and 140 RPM

Day 1: Preconditioning

• Prepare n+1 flasks with 10ml LB
• Transfer 10uL of each inoculated culture into their respective flask
• Put all flasks in the incubator and incubate overnight at 30° C and 140 RPM.

Day 2: Transformation

• Prepare 3n+2 test tubes with 1ml LB for three replicates per strain, as well as blank and –DNA.
• Put 100ng of the Kanamycin resistant AB gDNA into all tubes except the two designated for the blank and the –DNA control
• For each preconditioned flask from Day 1, transfer 70uL of each flask into three separate tubes containing 1mL of LB and the DNA (this will give us three trials for each strain we are trying to measure competence for)
• Add 70uL of one of the strains to the –DNA control tube (this ensures that the antibiotic is effective, and can be determined the day after plating)
• Incubate overnight at 30° C and 140 RPM

Day 3: Dilutions and Plating

• Obtain test tubes containing 10mL of sterile saline solution (get three times as many tubes as there are test tubes undergoing transformation in the incubator)
• Create a dilution series of 1:100, 1:10000, and 1:1000000 (this is done by transferring 100uL of the transformed culture to the first saline tube, vortex to ensure mixture, transfer 100uL of this into the second saline tube, vortex, then transfer 100uL of this into the third saline tube, vortex)
• Plate 100uL of each 1:100 dilution on plates of LB + Kanamycin
• Plate 100uL of each 1:1000000 dilution on plates of LB only
• Incubate overnight at 30°C

Day 4: Plate counting

• Start by ensuring that the antibiotic is effective (there should be no growth on the –DNA control plate that contains LB+Kan, but growth on the LB only plate is expected
• If antibiotic effectiveness is confirmed, count all colonies on each plate and record the results

-- Main.AurkoDasgupta - 06 Aug 2012

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Topic revision: r1 - 06 Aug 2012 - 19:37:55 - Main.AurkoDasgupta
 
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