Sanger Tree of Life HMW DNA Extraction: Automated MagAttract v.2

Prepare a lysis buffer master mix:

A	B
Phosphate-buffered saline (PBS)	200 µL
Proteinase K	20 µL
RNase A	4 µL
Buffer AL	150 µL

Set a heat block to 25 °C.

For samples which have been cryogenically homogenised:

1. Transfer 25 mg of the sample into a 2 mL microcentrifuge tube, then hold on dry ice to keep the sample frozen.
2. Add 374 µL of the lysis buffer master mix to sample, then homogenise the sample and master mix by gently pipetting 10 times with a wide-bore pipette tip.

For PowerMashed samples (weight less than 25 mg):

1. Transfer sample into a 1.5 mL BioMasher II tube and add 374 µL lysis buffer.
2. Disrupt sample in lysis buffer using the Diagnocine PowerMasher II tissue disruptor and BioMasher pestle, until no large pieces remain or sample cannot be disrupted further. (For more detailed instructions regarding PowerMashing, please refer to the Sanger Tree of Life Sample Homogenisation: PowerMash protocol.)
3. Transfer the entire contents of the BioMasher tube to a 2 mL microcentrifuge tube using a wide-bore tip.

Centrifuge sample tubes briefly and incubate on the heat block at 25 °C for 2 hours.

While samples are lysing, 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 briefly centrifuge to spin down.

Using a wide-bore pipette tip, set the volume to 380 µL, transfer lysate from the sample tubes to individual wells in the sample plate, taking care not to transfer large pieces of debris if possible.

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.