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Protocols in the laboratory

Before phylogenetic research can start, DNA of the organisms under investigation have to be collected. Protocols for extracting (fungal) DNA can be found here and protocols describing PCR, agarose gels preparation and electrophoresis, restriction fragment length polymorphism (RFLP), sequencing with BigDye 3.1, amplified fragment length polymorphism (AFLP), reverse line blotting (RLB), rolling circle amplification (RCA), loop-mediated amplification (LAMP).

dna structuur

Before starting with one of the molecular-biological techniques (PCR, sequencing, RFLP, AFLP, whole genome sequencing, qPCR), it is necessary to obtain good genomic DNA of the organism.

DNA extraction using CTAB

pcr-machine

PCR machines can be found in all kinds of sizes and brands, but in the end, they do all the same thing.
A PCR is carried out to amplify a gene or part of a gene. This amplicon can be used for downsstream experiments, like AFLP, RFLP or sequencing.

Executing a PCR reaction

PDF-files for most of the techniques can be downloaded here.

DNA extractions

Quick CTAB DNA extraction gfx0611
DNA extraction by Möller

PCR

Performing a PCR (50 µl reaction)

Master mixtures (25 µl reaction)

A test was done with one year old cultures (dried out). The old fungal tissue was used for DNA extraction using the 'Quick CTAB' method. Before extraction the tissues was also pre-incubated on liquid medium for 1 day. During extraction proteinase K as well as PVP (polyvinylpyrollidone) was used. The result of these extractions and the PCR of ITS1-ITS4 (ribosomal DNA) and Bt2a-Bt2b (beta-tubulin) is visible in the PDF files.

PCR condities