Part:BBa_K2429033:Experience

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Applications of BBa_K2429033

ASOs are unstable in the cell. Therefore, we needed to ensure the proper ASO to reporter ratio. We suspected that our system would be able to affect the splicing of mKate up to a threshold, after which the mKate would saturate the 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-ff4 from 10 to 500 ng using ASO3 and ASO2+. The ASO2+ was co-transfected with Ms2, so that the Ms2 could bind to the hairpin loop attached to the ASO+. (For a detailed explanation of how to plan a mammalian transfection click here)

mKate Titration for ASO3

mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for ASO3. The color of the line indicate the transfection bins of each result.

For the mkate titration for ASO3, we observe a standard mkate curve for all reporter concentration above 100 ng. Below 100 ng there was a disturbance in the normal curve, indicating that our system might be acting on the system.

mKate Titration for ASO2+

mKate-ff4 reporter amounts (10ng-500ng) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

For the mkate titration for ASO2+, we observe a disturbance at 100 ng of reporter. This indicates that our system is affecting the red output at this concentration. While expected to see a consistent output of red below our saturation threshold, the results still hold for the 100 ng case.

Based on these results, we decided to transfect with 100ng of mkate-ff4 moving forward.

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