We finally have probes

We reran our probe synthesis PCR to replace the control probes that had evaporated in the thermocycler during the last round. This time we were successful in getting both the DIG- labeled probe and the unlabeled control to show up properly on the gel run. This was my first time actually running a gel on my own, and I learned an important lesson: you must press the start button or nothing happens. It seems pretty obvious, but my assumption that the voltage box started automatically was quite incorrect. The other thing I learned was how to pour the tiny slide gels, and it’s not as daunting as others had made it out to be. If a little gel goes over the slide edge initially but the whole slide is still covered evenly, it’s okay to cut it off with a straight spatula edge once it solidifies.

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The bands are faint in the light, but they are there. From left to right: ladder, labeled probe, unlabeled control.

The gel run showed two bright bands in slightly different positions, which was exactly what we were looking for. The control probe is a little smaller and lighter because it has nothing attached to the DNA backbone, so it travels at a faster rate down the gel and comes out slightly ahead of the labeled probe.

Drew is happy for us getting the kinks worked out of this step, but her hesitation before proceeding is that we have no confirmed pathogen-negative stars to get tissue from (even the asymptomatic ones sometimes show amplification of the putative pathogen’s DNA). To strengthen the argument that this is pathogen DNA binding to the tissue samples, Drew suggests we extract DNA from sea star species that seem to be unaffected by wasting disease and make sure that the pathogen DNA does not amplify in those samples. This would be more convincing that we have presence of pathogen instead of some non-specific binding to something present in the caecum of all sea stars regardless of disease.

 

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