184.108.40.206. DNA extraction
For rapid DNA analyses, including genotyping with the markers described below, an extraction step is necessary:
- Place one leg from an adult SHB into a 1.7 ml centrifuge tube.
- Grind with a disposable pipette.
- Add 200 µl of 5% Chelex®-100 solution (Bio-Rad Corp.; Walsh et al., 1991).
- Vortex the solution.
- Incubate for 15 min at 95°C and 30 min at 55°C before cooling.
- Remove aliquots of 1 µl from this
solution, which will suffice for genetic tests.
When removing these aliquots, care must be taken to avoid including the Chelex® resin, as this can inhibit the next steps.
Alternate extraction methods for long-term use of DNA (e.g. the ‘CTAB’ method and the commercial DNEasy kit from Qiagen) are also effective and are described in detail in the BEEBOOK paper on molecular methods by Evans et al., (2013). Extractions of legs are advised since this avoids the heavily pigmented elytra, which can lead to inhibition of enzymatic assays and procedures.