Resource pcr purification kit

Amplification of the partial mtCO1 fragment was performed using forward primer 2195Bt (5ʹ-TGRTTTTTTGGTCATCCRGAAGT-3ʹ) and reverse primer C012/Bt-sh2 (5ʹ-TTTACTGCACTTTCTGCC-3ʹ) (Mugerwa et al. 2018 ). PCR reaction mixtures (20µL) contained 10µL of 2 × reSource™ Taq Mix (reSource Taq DNA Polymerase, 6 mM MgCl₂, 2 mM dNTPs) (Source BioScience, UK), 1µL of each 10 µM primer, 6µL of molecular biology-grade water (Sigma-Aldrich) and 2µL of DNA template. Initial denaturation (94 °C 2 min) was followed by 35 cycles of denaturation (94 °C, 20 s), primer annealing (52 °C, 30 s) and extension (72 °C, 1 min). A final extension (72 °C, 10 min) was performed before storing reactions at 4 °C. Electrophoresis of PCR products was on 2%(w/v) agarose gels in 0.5 × TBE stained with RedSafe™ (iNtRON Biotechnology, Korea). PCR products were visualised under UV light (302 nm) and those of the expected size (864 bp) purified for sequencing and cloning using a reSource™ PCR purification kit (Source BioScience, UK). Purified PCR products were sent for Sanger sequencing (Source BioScience, UK). Where a novel sequence was identified, purified PCR products were cloned from three separate PCR reactions using the pGEM®-T easy vector kit (Promega, UK) and resequenced to confirm the novel sequence. Sequences generated were deposited in GenBank (accession numbers MK444227-MK445130).

Amplification of a partial fragment (867 bp) of the mtCO1 gene was performed using the forward primer 2195Bt (5′-TGRTTTTTTGGTCATCCRGAAGT-3′) in combination with a reverse primer C012/Bt-sh2 (5′-TTTACTGCACTTTCTGCC-3′) [1 (link)]. PCR reaction mixtures (20 µL) were set up containing 10 µL of 2 × reSource™Taq Mix (reSource Taq DNA polymerase, 6 mM MgCl₂, 2 mM dNTPs, enhancers and stabilizers) (Source BioScience, Nottingham, UK), 1 µL of each 10 µM primer, 6 µL molecular grade water and 2 µL DNA template. A negative control (molecular biology grade water used in the place of DNA template) was included in every PCR run. PCR cycling was performed in a 2720 Thermal cycler (Applied Biosystems, Foster City, CA, USA) programmed as follows: Initial denaturation at 94 °C/2 min, 35 cycles of 94 °C/30 s, annealing at 52 °C/30 s and extension at 72 °C/1 min, with a final 10 min elongation cycle at 72 °C. PCR products were visualized through stained agarose gel after electrophoresis under ultra violet (UV) light. Amplified PCR products of expected size were purified using a resource PCR purification kit as per manufacturer’s instructions (Source BioScience, UK). Purified PCR products were sent for Sanger sequencing at Source Bioscience (Nottingham, UK).