Mangotaq colored reaction buffer

The species M. guilliermondii and R. mucilaginosa, isolated on SDA from a human toenail affected by onychomycosis were molecularly confirmed targeting the Internal Transcribed Spacer using the ITS1 and ITS2 primers [32 ]. The DNA extraction was performed using the commercial kit Animal and Fungi DNA Preparation Kit^® (Jena Bioscience, Jena, Germany) according to manufacturers’ protocol. The PCR (Polymerase chain reaction) amplification took place in a 25 µL final volume of mixture reaction containing: 1X MangoTaq Colored Reaction Buffer (Bioline, London, UK), 2.5 mM MgCl₂ (Bioline), 0.5 mM dNTP (Bioline), 0.5 mM of each primer (Macrogen Inc., Seoul, South Korea), 1.25U/µL MangoTaq (Bioline) and 2 µL of DNA. The amplification series consisted of 35 cycles of the following: 95 °C for 30 sec, 56 °C for 30 sec and 72 °C for 30 sec.

The DNA of the R. mucilaginosa isolate obtained on SDA control media was extracted using the Animal and Fungi DNA Preparation Kit^® (Jena Bioscience) according to the manufacturers’ instructions. The ITS1 and ITS2 primer sets targeting the ITS (Internal Transcribed Spacer) region were used for identification⁴⁶ . For PCR amplification, the following mixture was used in a 25 µL final volume: 5 µL of 5X MangoTaq Colored Reaction Buffer (Bioline), 1.25 µL of 50 mM MgCl₂ (Bioline), 0.5 µL of 10 mM dNTP (Bioline), 1.25 µL of each primer (Macrogen Sequencing Service, Korea), 0.25 µL of 5 U/µL MangoTaq (Bioline) and 2 µL of DNA. Negative control samples were used for each pair of primers. The amplification series consisted of 35 cycles of the following: 95 °C for 30 s, 56 °C for 30 s and 72 °C for 30 s. The fragment obtained after amplification was sequenced at Macrogen (Macrogen Sequencing Service, Korea).