We reran our probe synthesis reactions this morning. Comparing reaction conditions of the different kits, we concluded they were essentially the same, and we should continue using our original probe synthesis kit. However, we decided that we should use our PCR product as the template DNA for the probes instead of the DNA extracted from the original tissue samples, the caecum of diseased P. ochraceus. This would give us a better chance of getting a probe after replication, because we were starting with a high concentration of the target sequence and little else. Our original extracted DNA, which we used for the PCR, was still highly concentrated but contained a lot of other sequences from the tissues, effectively diluting our target.
After waiting eagerly for the thermocycler to finish replicating our probes, we opened it to find that one of the caps on our controls (unlabeled probes) had not been closed tightly enough, so all the liquid had boiled out. Only a stiff film was left on the bottom which we could not suck up in a pipette. Since we were testing two primer sets, we hoped that the one set that was closed would be a winner and give us bright bands on the gel. This was not the case. We got a faint band for the labeled and unlabeled probe in primer set 1, and a super bright band from the labeled probes in primer set 2 without a control. Ruth and I are happy to finally be successful in generating a probe for the sea star primers, since it means in situ hybridization with our slides is within reach. However, since we don’t have any controls, we cannot use this set for the ISH. It’s easy enough to set up synthesis for another set of probes, but we’ll have to wait to see if the next set produces bright bands as well instead of remaining unlit like our previous efforts. Fingers crossed, we will have our complete set of probes by tomorrow.
As a side note, I really like making the gels on glass slides because they take less agrose, less buffer, and they are super portable if you need to move to a darker room to see things light up. On the downside, they require a very steady hand to load, and if you set it down flat on a glass surface, the surface tension of the wet underside will stick it to the glass and it won’t come off without a major effort.