PCR Memory Hook

by Rafael Flores & Manuel Mayorga Labtimes 06/2016

Rafael Flores and his technician, Manuel Mayorga, work at a plant clinic in Seville, Spain. They use Real-time PCR as a tool for detecting pathogens in plant disease diagnosis. Willing to get rid of some of the hurdles of this technique, they have invented a “Microplate Filling Cassette” that helps to prevent contaminations in PCR and facilitates pipetting.

Researchers familiar with Real-time PCR know that the filling of qPCR microplates is not an easy task. It is not only boring and tedious. The microplate wells are small, quite close together and numerous (96 in a standard microplate). Their loading requires careful pipetting. And if you have many samples and want to be careful, the microplate filling will take its time – and with time, problems will begin to grow. Pipetting errors increase, because it becomes easier to get distracted and lose the filling pattern (hey, wait for a moment... where was I?... E6, F6, E7...? – panic – E5, perhaps...?). You have to be really focussed on what you are doing, otherwise you get lost and confused among so many wells. But remaining focussed for so long is not always easy.

The prolonged pipetting process also increases the risk of contaminations (to which Real-time PCR is extremely prone). It makes little sense to leave all the microplate wells uncovered till the very last well is loaded and the microplate, ready for amplification, is sealed with a film. During the microplate filling progress, you may feel a bit frightened by all these open wells permanently exposed to contaminations coming either from the environment, the remaining sample tubes or the positive controls that you are manipulating, opening and closing next to the microplate.

A simple plastic sheet with a hole in the middle prevents contaminations during filling of PCR plates and helps to remember the right well.

Frightened about open wells

Trying to avoid these contamination risks, some researchers load their samples in a laminar flow cabinet, believing that these cabinets provide a safer environment or higher sterility in PCR. But these cabinets were made for other purposes and, in fact, they only prevent contamination by air-borne microorganisms, which the filters can retain. If the target of your PCR is not the DNA of such an organism, using the cabinet is a questionable decision. Furthermore, they are often uncomfortable and noisy, making the focussing effort even harder.

In some cases the air flow may accelerate the evaporation of reaction mixtures (very often consisting in just a few microlitres) or even mediate cross-contamination by dispersing microdrops or aerosols from tubes or pipette tips over the microplate. Some researchers even load the samples in the cabinet under a dim light, to prevent the inactivation of photosensitive probe dyes.

To get rid of all those concerns, we came up with the idea to keep the wells covered during the filling of the microplate. To this end, we designed a simple instrument, we called a “Microplate Filling Cassette”. It consists of a base that holds the microplate and a mobile lid with a central window (see photo above).

The lid covers all the wells of the microplate, except the one located just under the lid window during loading. This way, the instrument helps to prevent airborne, aerosol-mediated and cross-contamination in PCR.

And it has another great advantage: the lid window indicates, in which well you are pipetting, making pipetting much easier and less error-prone.

Relaxed filling with cassette

Summing up, the cassette enables safer and more relaxed filling of microplate wells. You may be distracted or even interrupt the filling for a while, without a problem: the wells are covered and protected from contamination and evaporation, and the lid window shows, at which well you have to resume your work.

Our invention has been patented and we are looking for companies interested in its production and commercialisation. For more information visit www.microplatefillingcassette.com.

Last Changed: 28.11.2016