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Team:Alberta/plasmidconstruct
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<P>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 <i>E. coli</i> 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). </p> | <P>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 <i>E. coli</i> 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). </p> | ||
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<p><b><a href="####">pKan Sequence</a></b></p> | <p><b><a href="####">pKan Sequence</a></b></p> | ||
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<h2>Future Plasmid Construction</h2> | <h2>Future Plasmid Construction</h2> | ||
| - | Although our time was limited, we still wish to further validate the BioBytes assembly method by constructing longer plasmids. The next construct is planned to contain an artificial operon composed of 2 resistance markers ORFs and a red fluorescent protein ORF. | + | <P>Although our time was limited, we still wish to further validate the BioBytes assembly method by constructing longer plasmids. The next construct is planned to contain an artificial operon composed of 2 resistance markers ORFs and a red fluorescent protein ORF. |
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Revision as of 23:14, 21 October 2009
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Plasmid Construction on a BeadThe first plasmid to be designed and built using the BioBytes system was named pKan. The overview of the construct is shown (Figure 1). Figure 1.
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). Figure 2.
From this digestion, we observe that 8/10 of the screened colonies are the expected length. This was confirmed by sequencing. The sequence results can be viewed: Future Plasmid ConstructionAlthough our time was limited, we still wish to further validate the BioBytes assembly method by constructing longer plasmids. The next construct is planned to contain an artificial operon composed of 2 resistance markers ORFs and a red fluorescent protein ORF. |
