We grew monocultures of uninduced and induced wild-type CAT-I along with induced monocultures of the S27M and Q30M mutants following the monoculture growth assay outlined previously. We utilized the Qiagen Large-Construct Kit following the protocol for high yields of large-construct DNA without the removal of genomic DNA. The traJ and traI genes were PCR amplified using 2X GoTaq Green MasterMix starting from 10 ng of template from each sample. PCR products were loaded on a 1% TAE gel containing ethidium bromide. Bands were visualized under UV light and imaged with Carestream Gel Logic 112.
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Monoculture Growth and Plasmid Isolation
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Corresponding Organization : Johns Hopkins University
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Variable analysis
independent variables
- Monoculture growth conditions (uninduced wild-type CAT-I, induced wild-type CAT-I, induced S27M mutant, induced Q30M mutant)
- Presence and size of the traJ and traI genes as observed on a 1% TAE gel
- Amount of template DNA (10 ng) used for PCR amplification
- PCR protocol (2X GoTaq Green MasterMix)
- No positive or negative controls were explicitly mentioned in the input protocol.
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