Team:Sorbonne U Paris/Demonstrate


Our aim on part 1 is to show how we determined whether we succeed a cloning or not. Then on part 2 we will explain how we intend to demonstrate that our SugaRevolution system does work.

1. Determination of cloning succes

Here is an example of our cloning experiments. You can see below the photo of the gel on which all our first cloned and digested fragments have migrated.

Figure 1 : Migration of checking ligation. This two pictures above come from the same gel, but the black one is overexposed to show the tiniest band.

After we have made our first level 0 cloning, we digested them with the adequate digestion enzyme, cutting next to the insert sites, here BsaI. If the product of the digestion had the expected length we would have collected them, using gel to pcr DNA purification, and would have sent our DNA samples to sequencing. In our case the DNA was not of the correct length, probably because the sequence cloned was not the right one or because something went wrong while cloning. Then, we performed the cloning procedure once until it works..

In the picture above (figure 1) you can see that Paro1 (0.8kbp), Paro 2 (0.8kbp), 3’LTR (0.02kbp), 5’3 LTR (0.07kpb) having the expected length. We sent 3’2 and 3’4 (3’LTR), paro 1, paro 2 and 5’3 LTR to sequencing. Gag/Pol 3 and 5 were the only Gag/Pol subsequences that had the expected length (4.3kbp). These were also sent to sequencing.

To have an otherview of our progression in cloning, please refer to the results page. Precision on parts that were cloned and on their sequence quality are explained there.

The project is still now on progress. Indeed, cloning experiments and assessment of our retrotransposon activity in Chlamydomonas are conceived and planned for weeks to come.