Sanger Tree of Life HMW DNA Extraction: Plant Organic HMW gDNA Extraction (POE)

Prepare an adequate volume of the ‘Direct Plant Lysis Buffer’ (recipe in Materials).

• Preheat the direct plant lysis buffer for 15–30 mins, 65 °C at 400 rpm prior to use, ensuring the total dissolution of reagents.
• Add DTT and Proteinase K to the direct plant lysis buffer immediately prior to use, ensuring both reagents are thoroughly mixed.

Aliquot 65–100 mg of cryogenically disrupted tissue samples into individual 2 mL Lo-Bind tubes on dry ice.

Transfer 2 mL Lo-Bind tubes containing sample to wet ice for 10–15 minutes, allowing sample temperature to equilibrate.

Perform the direct sample lysis.

Add 550 μL of preheated direct plant lysis buffer (65 °C) to the first sample, immediately pulse vortex 5 times at full speed and place on a heat block at 55 °C, 600 rpm. Repeat for each sample.

Once all samples are homogenised and have began incubation, inspect each by inverting to mix. Any samples with aggregated tissue that can’t be homogenised through inversion should be mixed with wide bore p1000 until homogenous.

After 15 minutes of incubation, add 4 µL RNase A to each sample and mix by inversion until any aggregated, insoluble or sedimented tissue particles are resuspended. Repeat step 4.2 for severely reaggregated samples.

• The 4 µL RNase A can be diluted in 6 µL PBS per sample and added to the sample with a Multipette to improve ergonomics.

Incubate for another 45 minutes, 55 °C at 600 rpm.

• Do not agitate by mixing for the last 15 minutes of lysis; allow any unlysed sediment to settle at the bottom of the tube.

Whilst the samples are incubating, prepare fresh 2 mL Lobind tubes containing 150 μL of cold potassium acetate (4 °C; 5 M; pH 7.4) for each sample, and place on wet ice.

Remove the samples from the heat block, allow the lysate to settle for 5 mins at RT, and centrifuge for 10 minutes, 8,000 rpm at room temperature.* Avoid disturbing the settled insoluble tissue prior to centrifugation.

Use a wide bore p1000 to transfer the supernatant to its corresponding 2 mL Lo-Bind tubes containing 150 μL cold potassium acetate (4 °C; 5 M; pH 7.4), and carefully mix by pipetting with the same wide bore until homogenous.* gDNA is highly susceptible from this point; handle samples with care.

• The precipitate should appear whitish, opaque and slightly viscous.

Incubate the samples on wet ice for 5 minutes (precipitated samples can be left on wet ice for up to an hour if a break is required).

500 μL of Sera-Mag™ Speedbead solution and 175 μL of AMPure® PB beads per sample should now be removed from the fridge to equilibrate to room temperature.

A tabletop centrifuge should now be pre-chilled to 4 °C.

Perform the first chloroform separation (C:IA) in the fume hood:

Add 700 μL cold chloroform:isoamyl alcohol (–20 °C; 24:1, v/v) to the samples.

Mix on a tube rotator at 25 rpm for 10 minutes.

Centrifuge at 13,000 rpm for 5 minutes at 4 °C.

Transfer up to 700 μL of the aqueous phase (top layer) to a fresh 2 mL Lo-Bind tube using a wide bore p1000.

• Carefully aspirate from the top of the aqueous phase to avoid ’dragging’ contaminants from the interphase into the pipette.

Perform the second chloroform separation (C:IA) in the fume hood:

Add 700 μL cold chloroform:isoamyl alcohol (–20 °C; 24:1, v/v) to the sample.

Mix on a tube rotator at 25 rpm for 10 minutes.

Centrifuge at 13,000 rpm for 5 minutes at 4 °C.

Transfer up to 600 μL of the aqueous phase (top layer) to the applicable empty well of the ‘Sample Plate’ (See step 12) using a wide bore p1000.

• Carefully aspirate from the top of the aqueous phase to avoid ’dragging’ contaminants from the interphase into the pipette.

Add 500 µL Sera-Mag™ SpeedBead solution to the well containing the sample of the ‘POE Sample Plate’.

Label seven KingFisher™ 1 mL 96-well Deep-well plates and one KingFisher™ 200 µL standard 96-well plate with the following labels, and fill all applicable wells of each plate with their corresponding reagents (see table below).

A	B	C
POE Tip plate 1	1 mL	96-well tip comb (no reagent)
POE Sample plate	1 mL	Up to 600 μL aqueous phase of sample + 500 μL Sera-Mag™ Speedbead solution
POE Ethanol wash 1.1	1 mL	1 mL 80% ETOH
POE Ethanol wash 1.2	1 mL	1 mL 80% ETOH
POE Elution Plate 1	1 mL	400 µL Buffer EB
POE Tip plate 2	1 mL	96-well tip comb (no reagent)
POE Ethanol wash 2	1 mL	1 mL 80% ETOH
POE Elution Plate 2	200 µL	135 µL Buffer EB

Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex POE Protocol Script/attached KFX file in the Materials section) and select using the play button.

Load the filled plates onto the instrument following the instructions provided on screen and initiate once ready.

The instrument will prompt once the 1X SpeedBead SPRI is finished: add 175 µL (0.45X) AMPure PB beads to each well containing sample of the ‘POE Elution Plate 1’, place the plate back into the instrument, and continue the run.

The instrument will prompt when the 0.45X AMPure PB SPRI is finished: remove the ‘POE Elution Plate 2’ and use a wide bore p200 to transfer the 135 µL sample eluate to an appropriate tube for gDNA storage.

Incubate the sample at RT overnight to allow the gDNA to solubilise.

Proceed to appropriate QC checks and downstream processing.

Store the DNA at 4 °C.