DNA extraction for adults and immature stages.
DNA should ideally be obtained using relatively non-destructive techniques, to ensure that a voucher specimen is available for future morphological examination (Floyd et al. 2010).
For fruit fly adults 1-3 legs or the head of a specimen can be used, therefore retaining many other valuable morphological features of the specimen (such as wings, thorax and abdomen).
For larvae, anterior and posterior sections can be sectioned off and retained, preserving the morphologically valuable mouth parts and spiracles.
For aged, dried or generally degraded fruit fly adults, destructive sampling may be required. In this instance, we recommend taking high quality diagnostic images to retain morphological information for future examination.
There are many DNA extraction kits available, however the most commonly used are:
Preparation of samples
|Sample type||Suggested preparation|
|Adult fruit flies/larvae||If removing specimen from ethanol dry on blotting ‘tissue’ until ethanol has evaporated|
|Eggs||Usually require multiple eggs (>5) to be processed together to obtain enough DNA. However, 1-2 eggs are sufficient for use in the real-time PCR assay.|
|Aged, dried specimens||Destructive sampling is recommended to obtain enough DNA. Ensure sample is well homogenised using either a bead-mill (1 min @ 30 MHz) or with a plastic micro pestle in a microfuge tube.|
Modifications of kit protocols to increase DNA concentration
|Step||Advised protocol||Suggested modifications|
|First incubation step||Heat block or water bath at 56°C||Incubate overnight at 37°C. Add an additional 10 µL of proteinase K half way through incubation for immature stages and aged/dried samples.|
|Final elution step||Add 100 µL of elution buffer and incubate at room temperature for 1 min. Repeat step.||50 µL of elution buffer should be used for DNA to be used in the real-time PCR protocol. For empty pupal cases, or aged and dried samples, the total volume of the elution buffer can be reduced to 50 µL to increase the concentration. In addition to this, such samples should be incubated at 56°C for 5 minutes prior to centrifuging.|