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 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

adobe32Quick CTAB DNA extraction gfx0611
adobe32DNA extraction by Möller


adobe32Performing a PCR (50 µl reaction)

adobe32Master 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 conditiesexcel

Test results:

adobe32PCR with ITS1 and ITS4 primers on old cultures

adobe32PCR with Bt2a and Bt2b primers on old cultures 


Gel electroforese

adobe32Agarose gel electrophoresis  

Restriction fragment length polymorphism -
Amplified ribosomal DNA restriction analysis

adobe32RFLP or ARDRA


Amplified Fragment Length Polymorphism (AFLP) DNA-helix

adobe32AFLP op de genetic analyzer 310


adobe32Sequencing on ABI 37xx BigDye 3.1

Rolling Circle Amplification (RCA)

adobe32Rolling Circle Amplification

Reverse Line Blotting (RLB)

adobe32Reverse Line Blotting

Loop-mediated amplification (LAMP)

adobe32Loop-mediated amplification

More information about LAMP can be found on the EIKEN Genome Site.

An example

Multiplex Ligation-dependent Probe Amplification (MLPA)

adobe32MLPA One Tube Reaction manual

adobe32Optimizing MLPA fragment separation

adobe32Depurination of sample DNA

Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) 

MALDI-ToF principles