Quickextract extraction solution

Genomic DNA was isolated using QuickExtract Extraction Solution (Epicentre), and the frequency of endogenous gene disruption was evaluated using the Surveyor nuclease assay (Transgenomics) as described [36] (link). The CCR5 and BMPR1A genes were amplified by nested PCR using the Expand High Fidelity Taq System (Roche) and cloned into the plasmid pUC19 with the restriction sites EcoRI and BamH1. Sequence analysis was performed on individual cloned transformants as described [29] (link). Primer sequences are provided in Table S2.

The larva collected in 2016 was identified via the sequencing of a fragment of the mitochondrial nad4 gene in addition to morphological identification. DNA extraction (QuickExtract Extraction Solution, Epicentre, Madison, WI, USA) was performed according to the manufacturer’s protocol and the extracted DNA was kept frozen at -20 °C until further use.
A region within the nad4 sequence was amplified by PCR [29 (link)] using the following reagents: 5× Q5 OneTaq Standard Reaction Buffer (New England BioLabs, Ipswich, MA, USA; final concentration: 1×), 1.25 U of OneTaq Hot Start DNA Polymerase (New England BioLabs), 200 μM each of dNTP (‘dNTP-Mix 25 mM’, Biozym, Hessisch Oldendorf, Germany) and the primers N4J-8502D and N4N-8944D in final concentrations of 0.3 μM.
An initial denaturation (30 s at 94 °C) was followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 53 °C for 45 s and elongation at 68 °C for 60 s. The cycling ended with a final elongation step at 68 °C for 5 min.
After checking the size of the PCR product via agarose gel electrophoresis (2%, pre-stained with MidoriGreen, Biozym) the amplicon was prepared for sequencing using the Monarch PCR & DNA cleanup-kit (New England BioLabs) following the manufacturer’s protocol. Sequencing was carried out by Eurofins Genomics (Ebersberg, Germany).