Standard techniques for molecular biology such as cloning, PCR, immunochemical methods and others are established in our institute. Furthermore biochemical methods, like western blot analysis, SDS PAGE and protein expression are state of the art in our laboratories.

Currently we are working on cloning of different subunits from TRP proteins and diverse factors that are thought to influence Ca²⁺ signalling.

What is DNA?

Desoxy - ribonucleic - acid (short: DNA) is the bearer of the genetic information throughout heredity. This information is present as the genetic code, inscribed into the DNA.

The structure of DNA was enlightened in 1953 by James Watson and Francis Crick, who received the Nobel Prize of medicine in 1962 for their discovery.

DNA is a molecule consistent of different components, the so - called desoxy-ribo-nucleotides. Every nucleotide is an assembly of a sugar, a nucleobase [Adenin(A), Guanin(G), Cytosin(C) and Thymin(T)] and a phosphor - acid molecule. The structure of DNA is based on two nucleotide strands, which are arranged in the opposite direction and are helical winded through an intended axis (double - helix - structure). Defined sections on the DNA are referred to as genes. The sequence of the nucleobases is unique for every individual!

Methods:

Different strains of bacteria (Escherichia coli) serve as a tool to express DNA, which is cloned into several expression vectors, e.g. pEYFp-C1, which is an enhanced cyan fluorescent variant of the Aequorea victoria green fluorescent protein gene (GFP) which can furthermore be used for fluorescent microscopy.

Different subunits of Ca²⁺ channels are subcloned using restriction endonucleases, which are bacterial enzymes that cleave double-stranded DNA into defined fragments.

The fragment of interest can be obtained by electro elution and precipitated using alcohol and salt. DNA can be separated by an agarosegel through applying an electrical field. DNA migrates over a matrix on the basis of size; the negatively charged DNA flits to the positive pole and can be visualized through coloration with ethidium bromide that intercalates between stacked base pairs.

Through ligation into a designated vector and transformation into E.coli, recombinant clones can be screened using plasmid - minipreparartion. Clones that contain the Ca²⁺ channel subunit can be selected by antibiotic - resistance and restriction digests.

PCR - cloning

Another method for subcloning is called PCR (polymerase chain reaction). PCR is used to amplify DNA. Through addition of specific sequences (primers), DNA polymerase enzymes and applying of different temperatures DNA is amplified in a large amount.

Primers only bind to specific sequences within the target DNA and amplify only this special sequence of interest. This process can then be repeated for the resulting complementary strand resulting in the production of a copy of the original DNA present in the sample. With many cycles like this, DNA amplification can take place.

The different subunits of the TRP proteins are then used for patch clamp measurements and FRET experiments.