This is a modified protocol from the original MicroGEM PDQeX phytoGEM DNA extraction protocol provided by the manufacturer, see link here, and here.

The genomic isolation of Thuja plicata - Western redcedar

This protocol has been optimized to extract the DNA of Western redcedar leaves that have been dried with silica gel. This protocol is also suitable for lyophilised dry tissue.

Before starting

  • Turn on the PDQeX and start the UV DNA auto-cleaning (it takes 30min).
  • Add DNA-free water to Histoslv 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 to store the reagents in a cool place during the tissue disruption.
  • Every centrifuge step at room temperature (14-25C).
  • Completely thaw prepGEM and Histosolv, and mix by gently inverting the tubes. Remove 10XGREEN+ buffer and the Enhancer from the refrigerator and mix in the same way.
  • Prepare and label 24 2ml eppendorf tubes, 24 0.5ml tubes and x1 24-well plate or 8-strip tubes
  • Check there are CUT TIPS available, if not, get enough ready as shown

Caution

  • Do not load the PDQeX machine if the control screen indicates a temperature above 40° C.
  • Ensure the collection drawer and heating block are clean and DNA-free.
  • Ensure the collection drawer is inserted as far as possible, and that it is straight.
  • If fewer than 24 reactions are planned, make sure that the PCR tubes are placed in drawer wells corresponding to the channels to be used in the heating block

DNA extraction

  1. Samples selection Take the samples from their envelopes (24) and add ≤10 mg of dried tissue per sample by using a precision balance into a 2ml labelled eppendorf with two tungsten beads (3mm).
  2. Disruption of the samples Use the TissueLyser for 1min at 30Hz (x2, reversing the position of the adaptors). Placed 12 (or even number) samples per adaptor and be sure they are well balanced.

  3. Prepare the Extraction pre-mix:

    Reagent ul (x1 sample) ul (8x samples + 0.1) ul (24x samples + 0.1)
    10XGreen buffer 100 880 2640
    Histosolv 15 132 396
    Enhancer 10 88 264
    PrepGEM 2 17.6 52.8
    DNA-free water 10 88 264
  4. Mix the Extraction pre-mix by vortexing and spin it down.
  5. Pipette 137ul of the Extraction Mix into new 0.5ml labelled tubes.
  6. CAREFULLY discard the beads from the 2ml eppendorf tubes into a paper towel without loosing the tissue powder.
  7. By using a little spoon-spatula, collect the powder from each sample and add it to the 0.5ml tubes which contain the Extraction Mix. Clean the spoon-spatula with ethanol after every sample.
  8. Vortex the tubes thoroughly, don’t spin down.
  9. Transfer the solution (140ul), LIQUID and TISSUE into the PDQex catridges by using the CUT TIPS and put the cap on.
  10. Load 24-well plate or 8-strip tubes in the collection drawer and place it in the PDQeX.
  11. Insert the catridges on the top, flicking them before inserting in the heat block.

  12. Cover the cartridges with the hinged flap and close the sliding door.

    MAKE SURE THE PDQeX CARTRIDGES CORRESPOND WITH A COLLECTION TUBE OR WELL - OTHERWISE YOU WILL LOSE YOUR SAMPLE
    (this really happened to me)

  13. Select the plant programme and check the following conditions are set up:

    Temperature (C) Time (min)
    52 15
    75 10
    95 2
  14. Extract and discard the catridges.
  15. Take out the 24-well plate or the 8-strip tubes, seal it with a 36 Adhesive PCR plate sealer or with 8-strip caps.
  16. Check it’s properly labelled.
  17. 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.