Protocollen

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 extractions

Quick CTAB DNA extraction

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

Test results:

PCR with ITS1 and ITS4 primers on old cultures

PCR with Bt2a and Bt2b primers on old cultures 

Gel electroforese

Agarose gel electrophoresis  

Restriction fragment length polymorphism -
Amplified ribosomal DNA restriction analysis

RFLP or ARDRA

Amplified Fragment Length Polymorphism (AFLP)

AFLP op de genetic analyzer 310

Sequencing

Sequencing on ABI 37xx BigDye 3.1

Rolling Circle Amplification (RCA)

Rolling Circle Amplification

Reverse Line Blotting (RLB)

Reverse Line Blotting

Take a look at this illustrative video