Part:BBa_J100175:Experience

In one microfuge tube, we added 2uL (microliter) of 10X annealing buffer, 1uL of each of the 100 uM oligonucleotides (top and bottom), and 16 uL water to a total volume of 20 uL. The tube was boiled in approximately 500 mL of water for ten minutes and allowed to cool. Once cooled, we diluted the oligonucleotides one hundred fold and added 1 uL of the assembled oligos into the ligation.

1 uL of the promoter was added to the experimental tube (shown as J100175 in the figure), and 1 uL of the promoter was added to the negative control tube (shown as J110137). Each tube contained 9 uL of a master mix for Golden Gate Assembly, including the receiving plasmid J119137, BsaI, the ligase, water, and buffer.

We harvested colonies from each plate (experimental, labeled J100175 in the figure, negative control, shown as J119137, and positive control, J04450). Each sample was added to a microtube containing LBamp and grown overnight. We placed 200 uL of each into three wells and placed the media under UV light to measure fluorescence. We measured the ratio of fluorescence to cell density and averaged the three ratios for each sample. Then we took the standard deviation of those three ratios for our error bars.

This graph shows only the experimental promoter (J100175) and the negative control plasmid (J119137) to show the slight difference in values. The error bars were produced based on the standard deviation of the data.

This figure shows the experimental promoter (J100175), the negative control plasmid (J119137) and the positive control promoter (J04450). The positive control was known to produce Red Fluorescence, which accounts for the high values of fluorescence compared to the experimental and negative control. The error bars were produced based on the standard deviation of the data.

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