Part:BBa_K2429128:Experience

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Applications of BBa_K2429128

ASO Titration

Once we had determined a proper amount of mKate-ff4 plasmid, we wanted to check whether increasing the amount of ASO plasmid in the cell would knockdown our mKate levels further. We expected that as the amount of ASO increased in the system, more ASOs would be available to target the mKate-ff4. After a certain amount of ASO was added, the system would become saturated with ASO and the knockdown would level-off. The expected results can be seen below. The different colored lines corrispond to different transfection bins

In our experiment, we varied the amounts of ASO1 and ASO1+ from 20 to 200 ng. The ASO1+ 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)

ASO Titration for ASO1

ASO plasmid amounts (20ng-200ng) vs. the amount of red fluorescence (AU) for ASO1. The color of the line indicate the transfection bins of each result.

There is no substantial change across ASO concentrations.

mKate Titration for ASO1+

ASO plasmid amounts (20ng-200ng) vs. the amount of red fluorescence (AU) for ASO2+. The color of the line indicate the transfection bins of each result.

There is no substantial change across ASO concentrations.

In both results there was no decrease across the ASO levels. We concluded that 200 ng was not enough to have an effect on our system. Therefore, in further experiments we increased the levels of ASO to 300 ng and saw better results.

Based on these results, we decided to transfect with 300 ng of ASO moving forward.

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