Sanger Tree of Life HMW DNA Extraction: Automated Plant MagAttract v.4

Set one heat block with a 50 mL SmartBlock to 65 °C and another heat block with a 2 mL SmartBlock to 55 °C.

A	B
Phosphate-buffered saline (PBS)	200 µL
Buffer AL	150 µL

Prepare a lysis buffer master mix in a 50 mL centrifuge tube:

Place the lysis buffer on the 65 °C heat block and incubate at 400 rpm for at least 20 minutes. Keep at temperature until added to the sample.

Transfer 50 mg of cryogenically disrupted tissue from each sample to 2 mL microcentrifuge tubes.

• Ensure the disrupted tissue is completely disrupted into a fine powder; avoid matted/clumped powder. This is crucial for optimal DNA yield and integrity; poorly disrupted tissue drastically decreases lysis and extraction efficiency.
• Any samples containing poorly disrupted tissue ‘chunks’ should be flagged as requiring reprocessing and further cryogenically disrupted.

Transfer the samples to a pre-chilled cold block on wet ice and incubate for 10 minutes to equilibrate temperature.

Add 20 µL Proteinase K (for n+1 samples) to the preheated lysis buffer immediately prior to initiating lysis, swirling the centrifuge tube to mix.

Add 370 µL of the preheated lysis buffer plus Proteinase K to each sample, immediately homogenising the lysate by mixing with 5 rapid pulse vortexes, and place on the 55 °C heat block at 600 rpm for 15 minutes.

After 5 minutes incubation, resuspend any severely aggregated samples by pipette mixing with a wide-bore pipette tip.

After the initial 15 minute incubation, add 4 µL RNase A to each sample and mix thoroughly by inversion until any aggregated, insoluble or sedimented tissue particles are resuspended.

Incubate samples for a further 45 minutes on the heat block at 55 °C at 600 rpm.

During this incubation, samples should be occasionally mixed (every 5–15 minutes) by inversion to resuspend sedimented particles.Do not mix the samples by inversion for the final 15 minutes of lysis, allowing aggregated, insoluble or sedimented tissue particles to settle at the bottom of the tube.

Whilst samples lyse, label nine 1 mL 96-well deep-well KingFisher™ plates and fill the number of wells required for the number of samples in each plate as follows:

A	B
Tip plate	96-well tip comb (no reagent)
Elution 2	200 µL Buffer AE
Elution 1	200 µL Buffer AE
NFW Wash	500 µL nuclease-free water
PE Wash 2	700 µL Buffer PE
PE Wash 1	700 µL Buffer PE
MW1 Wash 2	700 µL Buffer MW1
MW1 Wash 1	700 µL Buffer MW1
Sample plate	15 µL Suspension G magnetic beads + 280 µL Buffer MB

Once samples have completed lysing, remove sample tubes from the heat block and allow the lysate to settle to the bottom of the tube for 5 minutes.

Centrifuge the samples for 10 minutes at 8,000 rpm at room temperature.

Using a wide-bore pipette tip, set the volume to 380 µL, gently transfer lysate from the sample tubes to individual wells in the sample plate, taking care to avoid aspirating the pelleted tissue particles.

Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex DNA Extraction 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.

Prior to loading the “Sample Plate”, the instrument will prompt to remove the “Tip Plate”. Once the final plate is loaded, the protocol will automatically begin; this takes approximately 50 minutes.

Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.

Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the DNA using a wide-bore pipette tip.

Using a 200 µL multi-channel pipette and wide-bore tips, pipette eluates from Elution Plate 2 into Elution Plate 1, and gently pipette mix 5–10 times with wide-bore tips to fully homogenise DNA in the eluate. Elution Plate 1 with the combined eluates is now the ‘Sample Plate’ for the 0.45X SPRI.

Set-up the KingFisher plates for the 0.45X SPRI as detailed below:

A	B	C
Tip Plate	1 mL Deep-well	96-well tip comb (no reagent)
Sample Plate (Elution Plate 1 from DNA Extraction Protocol)	1 mL Deep-well	380 µL DNA + 171 µL AMPure PB beads
Ethanol Wash Plate	1 mL Deep-well	1000 µL 80% EtOH (freshly made)
Elution Plate	200 µL standard	135 µL Buffer EB

Select the required 0.45X SPRI protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex 0.45X SPRI Protocol Script/attached KFX file in the Material section) and select using the play button.

Load the filled plates onto the instrument following the instructions provided on screen.

Once the final plate is loaded, the protocol will automatically begin; this will take approximately 40 minutes.

Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.

Using a wide-bore pipette tip, transfer the 130 µL of eluate from the elution plate into microcentrifuge tubes.

Incubate the DNA at room temperature overnight and perform the required QC the following morning.

Store the DNA at 4 °C.