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From 2009.igem.org

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- TNN, TTN, and TTL promoter constructs mutant at position 6 were ligated into the pGlow-TOPO vector (Invitrogen).

- Ligation products were transformed into TOP10 cells; cells were spread on antibiotic LB agar plates and allowed to grow at 37 °C overnight.

- Plasmids were isolated from cultures of TOP10 cells containing TNN, TTN, and TTL promoter constructs mutant at position 5. Reaction mixtures containing plasmid and a primer complimentary to the plasmid were prepared and submitted to the Biomedical Genomic Center at the University of Minnesota for DNA sequencing.

- 2 mL LB antibiotic cultures were innoculated from the DH5αPro cells transformed on 7-6 and allowed to grown overnight at 37 °C with shaking at 220 rpm.

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