Gotaq colourless hot start mastermix

Two separate PCRs were conducted on pooled DNA. To analyse the presence/absence of microsporidia within mosquito pools, samples were screened using primers 18f and 1492r of Ghosh and Weiss (2009) which amplify a region of the Internal Transcribed Spacer of the rDNA. PCRs were carried out using 1x GoTaq colourless Hot Start mastermix (Promega), 2µM each primer and 1µl DNA with a PCR profile of 95°C for 3 min then 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min with a final 5 min extension at 72°C. Confirmation of species identity of mosquito samples was established through mitochondrial DNA barcoding using the primers L1490 and H2198 of Folmer, Black, Hoeh, Lutz, and Vrijenhoek (1994) with a PCR mix of 1x GoTaq colourless Hot Start mastermix (Promega), 2µM each primer and 1µl DNA and a PCR profile of 95°C for 3 min then 35 cycles of 95°C for 1 min, 40°C for 1 min, and 72°C for 1.5 min with a final 5 min extension at 72°C. PCR products were checked by electrophoresis on 1.5% agarose gels then purified using a GeneJet PCR purification kit following the manufacturer's recommendations. Sequencing was performed by Eurofins Genomics (Konstanz, Germany). Samples from Pool D required dilution (1/10) prior to PCR due to coextraction of a PCR inhibiting compound.