This is a modified protocol from the original QIAGEN DNeasy Plant Mini Kit DNA extraction provided by the manufacturer, see link here

The genomic isolation of Pseudotsuga menziessi - Douglas fir

This protocol has been optimized to extract the DNA of Douglas-fir leaves that have been dried with silica gel. This protocol is also suitable for lyophilised dry tissue. Fresh samples can also be used, which may increase the DNA yield recovered.

Before starting

  • Turn on and preheat the water bath and set at 55C. When the temperature has bean reached, place in the AP1 Buffer.
  • Add ethanol to Buffer AW1 and Buffer AW2 concentrates if starting a new bottle.
  • Check where the tissuelyser adaptors are. If they are in the -80C freezer, take them out and rinse them with water, then leave them to dry.
  • Get some ice from the ancillary room (next door on the right from our lab).
  • Every centrifuge step at room temperature (14-25C).

DNA extraction

  1. Samples selection Take the samples from their envelopes (24) and add ≤20 mg of dried tissue per sample by using the precision balance in the small room, in a 2mL eppendorf with two tungsten beads (3mm).
  2. Disruption of the samples Use the TissueLyser in the small room, for 1min at 30Hz (x2, reversing the position of the adaptors). Placed 12 samples per adaptor and be sure they are well balanced.
  3. Put the 24 tubes in a big rack, leaving one epmty well between tubes to avoid cross contamination while pipetting. Add 400 μl Buffer AP1 (which has been placed in the water bath at the beginning to redissolve precipitates, do not mix it though). Add 20 μl of Protease K, which is stored in the 4C fridge in the main lab. Add 4 μl of RNase A.
  4. Vortex throughly and incubate for 1h at 55C in the water bath already pre heated.
  5. Pipette 130 μl of Buffer P3. Vortex and incubate at least 5min.
  6. Centrifuge the lysate for 5 min at 20,000 x g (14,000 rpm).
  7. Pipette the lysate (try to avoid touching the pellet of tissue created, if needed recentrifuge the samples tube) into a QIAshredder spin column placed in a 2ml collection tube. Centrifuge for 2 min at 20,000 x g.
  8. Transfer the flow-through into a new tube (no lid collection tube) without disturbing the cell-debris pellet if present. Add 675 μl of Buffer AW1, and mix by pipetting. Very important to mix it immediately one by one.
  9. Transfer 650 μl of the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge for 1 min at ≥6000 x g (≥8000 rpm). Discard the flow-through. Repeat this step with the remaining sample.
  10. Add 500 μl of Buffer AW2, and centrifuge for 1 min at ≥6000 x g. Discard the flow-through.
  11. Add another 500 μl Buffer AW2. Centrifuge for 2 min at 20,000 x g. Remove the spin column from the collection tube carefully so that the column does not come into contact with the flow-through.
  12. Transfer the spin column to a new 1.5mL microcentrifuge tube that has been already properly labeled.
  13. Add 50 μl of Buffer AE for elution. Incubate for 5 min at room temperature (15–25°C). Centrifuge for 1 min at ≥6000 x g. Repeat again.
  14. DNA is VERY FRAGILE, do not vortex it. To mix it flick it gently and spin down. Store it at 4C if it is going to be used soon (same week), store at -20C for long term storage.