It’s nice when you have a lot of people power to accomplish a lot of pippetting at once, but there is a point at which there are too many people in the small lab space to be effective. We found out that number is 4, if you’re not running around trying to fetch things. We were doing a preliminary test for primer binding so that we know we have reliable primers to attach probes for in situ hybridization. If the primers won’t bind, then we don’t want to attach probes because they will wash away when we try to hybridze them with DNA found in tissue samples taken from live organisms. The power of in situ hybridization is that it can detect viable DNA within an organism. Conventional PCR will only detect the presence of DNA, which may or be from a live organism (it could be dead or just within the body of the host organism and not doing anything).
The confusing part was that we have a bunch of different sea star and coral pathogen samples, all of which had primers that needed different temperatures. We did our best to consolidate them into four temperatures within a few degrees of ideal, with the addition of one touchdown PCR temperature cycle. Touchdown PCR is very interesting, since it allows you to work with primers for which you don’t know what the appropriate temperatures should be. It starts high and walks down by half a degree each cycle. The primers should start binding very specifically at its highest possible temperature, and then they bind nonspecifically the lower the temperature gets. However, by the time the temperature gets low enough to allow nonspecific binding, you should hopefully have a lot of specific binding primers that have replicated, multiplying the number of DNA copies enough to swamp out any nonspecific binding. It’s the ideal way to treat your PCR, because the reaction is not at the optimal temperature all the time, but it’s a good way to hedge your bets.
Learning how to program the thermocycler was also a bit of an adventure, but after figuring out how that everything had to be entered in seconds, it got easier, at least for the single block. The divided block was more difficult, since almost every program number we tried to assign was already taken, but in the end we managed it.