Sanger Tree of Life HMW DNA Extraction: Manual Nucleated Blood Nanobind®

Add 10–20 μL of nucleated blood to a 1.5 mL Protein LoBind tube.

Add 180–190 μL of 1 X PBS for a total volume of 200 μL.

Add 20 μL of Proteinase K.

Add 20 μL of RNase A.

Pulse vortex the blood sample 10 times for 1 second at the maximum setting, and then spin the blood sample tube on a mini-centrifuge for 2 seconds.

Incubate the blood sample at room temperature for 3 minutes.

Add 200 μL of Buffer BL3 and pulse vortex 10 times for 1 second at the maximum setting, then spin the blood sample on a mini-centrifuge for 2 seconds.

Incubate the blood sample on a heat block at 55 °C at 900 rpm for 10 minutes.

Pulse vortex the blood sample 3 times for 1 second at the maximum setting, and then spin the blood sample on a mini-centrifuge for 2 seconds.

Add a Nanobind disk to the blood sample tube.

Add 350 μL of 100% isopropanol to the blood sample and Nanobind disk.

Inversion mix the blood sample 5 times, then place the sample on a rotating mixer at 9 rpm for

15 minutes at room temperature.

Place the blood sample tube on the magnetic rack to allow for disk (and therefore HMW DNA) capture.

Discard the supernatant with a pipette, trying not to disturb the Nanobind disk or pipette the DNA. To minimise carryover contamination, remove any excess liquid in the tube cap.

Add 700 μL of Buffer CW1, then remove the blood sample tube from the magnetic rack, inversion mix 4 times, before placing the blood sample tube back on the magnetic rack and discarding the supernatant. To ensure that the Nanobind disk is captured towards the top of the tube, first place the tube in the rack separated from the magnet, then flip the rack so that the lid of the tube is on the benchtop. Add the magnet into the rack, then flip the now-assembled magnetic rack so that the sample is the right side up.

Add 500 μL of Buffer CW2, then remove the blood sample tube from the magnetic rack, inversion mix 4 times, before placing the blood sample tube back on the magnetic rack and discarding the supernatant. To ensure that the Nanobind disk is captured towards the top of the tube, first place the tube in the rack separated from the magnet, then flip the rack so that the lid of the tube is on the benchtop. Add the magnet into the rack, then flip the now-assembled magnetic rack so that the sample is the right side up.

Repeat step 16 for a total of two Buffer CW2 washes.

Spin the blood sample tube on the mini-centrifuge for 2 seconds, then place the blood sample tube back on the magnetic rack.

Remove any residual buffer that may be present in the tube; if the Nanobind disk is blocking access to the bottom of the tube to remove this liquid, it can be gently pushed towards the magnet with the tip of the pipette.

Repeat steps 17 and 18 to ensure that there is no residual buffer in the tube.

Remove the sample from the magnetic rack, then add 200 μL of Buffer EB directly onto the Nanobind disk and incubate the sample for 10 minutes at room temperature.

Transfer the eluate containing the HMW DNA to a new 1.5 mL microcentrifuge tube with a standard p200 pipette and 200 μL pipette tip. Repeat until all the eluate is transferred.

Spin the sample tube containing the Nanobind disk on a centrifuge at 10,000 × g for 15 seconds, then combine any additional liquid that comes off the disk with the previously transferred eluate.

Repeat step 23 for a total of 2 spins on the centrifuge if any visible DNA remains on the disk.

Pipette mix the eluate 10 times with a standard p200 pipette and 200 μL pipette tip to homogenise the DNA within the sample and disrupt any viscous regions.

Allow the eluate to rest overnight at room temperature to allow the DNA to solubilise.

Following the overnight rest, pipette mix 10 times with a standard p200 pipette and 200 μL pipette tip, then perform the required QC.

Store the DNA at 4 °C.