3.2.2 DNA extraction using Qiagen Blood and Tissue DNA kits
This is a reliable extraction method using a commercial kit sold by Qiagen (www.quiagen.com), it is suitable for honey bee guts, small larvae or tissues from larger larvae or adults (avoid using the compound eyes).
- Place 50 mg honey bee material in a centrifuge tube and mince thoroughly on ice with a mini pestle
- Add 180 µl Buffer ATL and 20 µl Proteinase K at the provided concentration
- Vortex 30 seconds and incubate at 56oC for 1 hour, vortexing for 30 seconds after 30 min
- Premix equal volumes of Buffer AL and ethanol (96-100%), mixing enough to provide 400 µl per sample plus 10% extra
- Vortex samples 30 seconds and add 400 µl AL/EtOH mix each, vortex again 30 seconds
- Pipette all into DNeasy Mini spin column nested in a 2 ml collection tube.
- Centrifuge at > 8000 rpm in a microcentrifuge (6k g). Discard flow-through and collection tube
- Place spin column in new 2 ml collection tube, add 500 µl Buffer AW1, centrifuge 1 min at > 8000 rpm. Discard flow-through and collection tube
- Place spin column in new 2 ml collection tube, add 500 µl Buffer AW2, centrifuge 3 min at > 14000 rpm. Discard flow-through and collection tube
- Remove spin column, checking to be sure ethanol is gone and place into a clean 1.7 ml centrifuge tube
- Add 200 µl Buffer AE to the centre of the
membrane, incubate at room temperature and then centrifuge for 1 min at >
8000 rpm. Eluted DNA will be in tube. Check quantity by Nanodrop or agarose gel
as in section 3.2.1 above.