Part:BBa_K187427:Experience

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Applications of BBa_K187427

This primer was used to amplify an origin of replication from pMB1. This origin was used with the Biobytes method to construct a plasmid consisting of the 75% promoter, a kanamycin resistance gene (BBa_K187021) and the origin of replication (using primers entered as BBa_187424 and BBa_K187425).

Once the linear chain was complete, an annealed terminator was added and the contruct was cleaved from the bead using USER. The sample was heated and cooled slowly to allow proper annealing of the ends before transformation. The anneal construct was transformed into E. coli and plated on Kanamycin containing LB plates. After growing overnight, we counted 105 colonies. Ten colonies were screened by digestion with I-SceI, which was expected to linearize the plasmid. The expected length of the linearized construct was 1595bp (Figure 2).

From this digestion, we observed that 8/10 of the screened colonies are the expected length. This was confirmed by sequencing.

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