Deep targeted DNA pool sequencing was used to search for genomic variation associated with insecticide resistance in each line. Genomic DNA was extracted from 2 batches of 50 adult females from each condition (F0 Guy‐R, F0LD80, F2LD25 and F2LD75, see Figure 6 ) using the PureGene kit (Qiagen) following manufacturer's instructions. DNA extracts obtained from each batch were quality‐checked on agarose gel, quantified using the Qubit dsDNA Broad Range kit (Qiagen) and mixed in equal quantity in order to obtain a single genomic DNA extract representative of 100 individuals for each condition.
Qubit dsdna broad range kit
Manufactured by Qiagen
The Qubit dsDNA Broad Range kit is a laboratory equipment product designed for quantifying double-stranded DNA (dsDNA) samples. It provides a sensitive and accurate method for measuring DNA concentrations within a broad range.
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3 protocols using qubit dsdna broad range kit
1
DNA Pooling for Insecticide Resistance
2
Amplification and Sequencing of TiLV
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Partial regions of the TiLV segment 1 genome were amplified by semi‐nested RT‐PCR as described previously (Taengphu et al., 2020 (link)). Five microliters of the second round PCR products were analysed by electrophoresis on a 1% agarose gel stained with ethidium bromide solution. The remaining 20 µl reaction volume from the second round PCR was purified for each sample on a NucleoSpin Gel and PCR Clean‑up column (Macherey‐Nagel) and eluted with 20 μl of the kit elution buffer (5 mM Tris‐HCl, pH 8.5). The purified products were quantified using Qubit dsDNA Broad Range kit (Qiagen) with a Qubit 3.0 fluorometer prior to Nanopore multiplex library preparation.
3
Partial TiLV Genome Amplification
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Partial regions of the TiLV segment 1 genome were amplified by semi-nested RT-PCR as described previously (Taengphu et al., 2020) (link). Five microliters of the second round PCR products were analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide solution. The remaining 20 µL reaction volume from the second round PCR was purified for each sample on a NucleoSpin Gel and PCR Clean up column (Macherey-Nagel) and eluted with 20 μL of the kit elution buffer (5 mM Tris-HCl, pH 8.5). The purified products were quantified using Qubit dsDNA Broad Range kit (Qiagen) with a Qubit 3.0 fluorometer prior to Nanopore multiplex library preparation.
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