Part:BBa_K2429017:Experience

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Verifying the construct

mKate Titration

Before being able to test our guides against our newly designed reporter, we needed to ensure it was exhibiting proper behavior. To that end, we ran an titration on the mKate-HBG construct. We expected that as we increased the amount of reporter in the system, the amount of mKate would rise, as the reporter was designed to output normally in the absence of our system. These expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of mKate-HBG from 10 to 500 ng. (For a detailed explanation of how to plan a mammalian transfection click here)

mKate Titration for mKate-HBG

mKate-HBG reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU). The color of the line indicate the transfection bins of each result.

The reporter is not producing red fluorescence above background noise.

Unfortunately, we were unable to test any of our constructs against this reporter, because the reporter was not producing red. '

Our next step is to debug our reporter. We suspect the problem may be with the intron we chose, or the way we built that intron, because the mKate-ff4 was outputting fine.

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