PCR

The Polymerase Chain Reaction (PCR) enables the amplification of DNA segments. Short DNA molecules, known as primers, serve as starting points for synthesis. In 25 to 35 cycles, the existing DNA is copied between the primer attachment points in each case, thereby doubling the number of molecules and resulting in an exponential amplification. 

Each cycle is made up of three different temperatures. First the DNA strands are separated (94 °C), then the primers are deposited (e.g. 55-65 °C, depending on the primer pairs) and finally the new synthesis of DNA takes place (usually 68-72 °C). 

Since the primers are contained in the products, this technique can also be used to introduce changes into the DNA. PCR can be used, for example, to amplify entire genes or parts of genes such as promoters and then incorporate them into microorganisms in new combinations.

PCR devices (thermal cyclers) are basically heating blocks in which the temperature profiles of PCR reactions can be run through particularly quickly.

Quantitative PCR

Quantitative PCR is a further development of PCR, in which the formation of the products can be followed during the reaction by means of fluorescence measurements. In the simplest case, a dye is added to a PCR reaction, which fluoresces in the presence of DNA (e.g. “Sybr Green”). By determining the fluorescence, the amount of DNA in the reaction vessels can be measured continuously. This makes it possible to infer the amount of starting (template) DNA and quantify it. This is hardly possible with a standard PCR, as only the amount of product at the end of the reaction can be determined.

By way of an example, qPCR allows the bacterial count of a species in a mixture of different organisms to be determined, or, in conjunction with reverse transcription, the quantification of individual RNA molecules (RT-qPCR).

In the Biochemistry laboratory, a LightCycler device (Roche) is used for this purpose. Here, the reaction takes place in small glass capillaries and the fluorescence is measured by rotating the capillaries one after the other in front of an optical system.

Electrophoresis

Agarose gel electrophoresis in particular is used for DNA analysis, in view of the fact that it enables the size of DNA molecules, such as those from PCR experiments, to be determined quickly and easily. After separation on the gel, the fragments are stained with ethidium bromide and photographed under UV light.

Media production

The media for the cultivation of the different microorganisms are sterilized by autoclaving. Depending on the culture, these must contain different nutrients. Selective media make it possible to identify cells containing certain DNA molecules, since only those can grow in the selective medium (which may contain an antibiotic, for example).

All culture residues containing genetically modified organisms are also autoclaved to prevent GMOs being released into the environment.

Cultivation of genetically modified microorganisms

Cultures can be treated in the safety cabinet under sterile conditions to prevent contamination by environmental microbes.

Incubators and various temperature-controlled shaking incubators are used to grow the microorganisms, providing ideal conditions for the microbes.

In addition, growth experiments can also be carried out in microtiter plates and a laboratory fermenter.