Neb monarch pcr and dna cleanup kit

Nucleic acid from homogenized mosquitoes was extracted individually, using the NucleoSpin DNA Insect Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany), following the manufacturer’s instructions. The mitochondrial COI region was amplified using the primer combination LCO1490 and UEA8 (targeting a ~1000 bp long fragment) [29 (link), 30 (link)], and PCR reactions were performed with the Platinum II Taq Hot-Start DNA Polymerase kit (Invitrogen, CA, USA), using 0.5 μl of the enzyme, 2 μl of 10x buffer, 0.6 μl of 50mM MgCl₂, 1–1 μl of the forward and reverse primer solutions (10 μM), 0.2 μl dNTP mix (Promega, USA), 14.7 μl nuclease-free water, and used 2 μl of the DNA extract as a template. The PCR protocol included 5 cycles with the following parameters: 30 sec at 94°C, 30 sec at 45°C, and 1 min at 72°C, followed by 35 cycles of denaturation for 30 sec at 94°C, annealing for 1 min at 51°C, and 1 min of elongation at 72°C, ending with a final extension step at 72°C for 10 minutes. PCR products were purified using NEBMonarch PCR and DNA Clean-up Kit (New England Biolabs, USA), following the manufacturer’s instructions. The sequencing was processed by Eurofins Genomics Custom DNA Sequencing service (Eurofins Genomics, Germany).