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).

  1. Place 50 mg honey bee material in a centrifuge tube and mince thoroughly on ice with a mini pestle
  2. Add 180 µl Buffer ATL and 20 µl Proteinase K at the provided concentration
  3. Vortex 30 seconds and incubate at 56oC for 1 hour, vortexing for 30 seconds after 30 min
  4. Premix equal volumes of Buffer AL and ethanol (96-100%), mixing enough to provide 400 µl per sample plus 10% extra
  5. Vortex samples 30 seconds and add 400 µl AL/EtOH mix each, vortex again 30 seconds
  6. Pipette all into DNeasy Mini spin column nested in a 2 ml collection tube.
  7. Centrifuge at > 8000 rpm in a microcentrifuge (6k g). Discard flow-through and collection tube
  8. 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
  9.  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
  10.  Remove spin column, checking to be sure ethanol is gone and place into a clean 1.7 ml centrifuge tube
  11. 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.