Part:BBa_K4683000:Experience

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Applications of BBa_K4683000

Lambert_GA 2022
Exponential Rolling Circle Amplification (eRCA) was successful with this part. The products of eRCA are short DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the exponential rolling circle products (eRCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, no/dim bands near the top of the well indicate that short DNA was produced(see fig. 1).

Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

By analyzing the results on the gel, our team concluded that short strands of DNA were produced, likely the eRCP.
The eRCP was also tested with DFHBI-1T dye as the RCP would consist of Lettuce Aptamer sequences. The fluorescence was read on the plate reader (see fig. 2).

Figure 2. Graph of fluorescence after DFHBI-1T was added

As seen in Figure 2, the increase in fluorescence of the eRCP+dye was significantly greater than the controls, which suggests that Lettuce aptamers were produced. According to these results, eRCA was successful.

User Reviews

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