Thermopol pcr buffer

A small, but reasonably range-extensive (see Table 1), sample of beetles harboring the most common mitochondrial haplotypes within lineage L1 (H1 [N = 28]; H2 [N = 21]) and L2 (H44 [N = 14]) (see Results) were surveyed for infection with the α-proteobacterium Wolbachia using a diagnostic PCR that targets the 16S rRNA of the bacterium [49 (link)]. This is a proven method with high specificity and detection rates [50 (link)]. In contrast to alternative approaches like nested-PCR (e.g., [51 (link)]), this method is not prone to environmental contamination and false-positive results. PCR was performed in 25 μL reactions containing 2 μL of DNA template, 1×ThermoPol PCR Buffer (NEB), 200 μM each dATP, dCTP, dGTP, 400 μM dUTP, 1 U Taq polymerase (NEB) and 0.8 μM each of the primers W-Specf and W-Specr [49 (link)]. The PCR cycling consisted of one cycle of 94°C for 2 min; followed by 40 cycles of 94°C for 30 s, 55°C for 45 s and 72°C for 1 min 30 s; and concluding with 10 min at 72°C. Wasps from an infected laboratory colony of Trichogramma pretiosum were used as positive controls. Amplification was checked by electrophoresis in a 1% agarose gel and the Wolbachia infection status of each specimen was assigned based on the presence/absence of a 438 bp diagnostic band.