Low-cost museum DNA extraction using magnetic beads

Lysis buffer C

200mM Tris pH8, 25mM EDTA pH8, 0.05% Tween 20, 0.4mg/ml PK

To make 10mL mix together: 7295µL molecular grade water, 2000µL 1M Tris pH8,

500µL 0.5M EDTA (pH 8), 200µL Proteinase K (20mg/ml), and 5µL Tween 20.

This is sufficient for 1 extraction plate of 95 samples and 1 negative control (90µL each).

Binding buffer D

5 M guanidine hydrochloride, 40% (vol/vol) 2-propanol, 0.12 M sodium acetate and 0.05% (vol/vol) Tween 20.

In a glass or plastic bottle, weigh 124.2g of guanidine hydrochloride and fill up with molecular water to 150mL. Heat briefly in a microwave until the buffer is warm to the touch and shake until the salt is fully dissolved. Add 104mL of 2-propanol, 10.4mL of 3M sodium acetate buffer solution (pH 5.2) and 130µL of Tween 20.

This is sufficient for 6 extraction plates of 95 samples and 1 negative control (450µL each). Store in a fridge for up to 4 weeks. Seal the bottle with Parafilm to avoid evaporation.

Wash buffer

Option 1: To 100mL of Qiagen buffer PE concentrate, add 400mL of 96-100% Ethanol.

Option 2: To 5mL 1M Tris-HCl, add 412mL 97% Ethanol and 83mL molecular grade water.

Note: Kevin Beentjes (Naturalis) has compared the Qiagen PE and "DIY" wash buffers with no apparent difference in fragment recovery.

This makes 500mL of wash buffer, sufficient for 6 extraction plates of 96 wells (750µL each). This buffer can be stored at room temperature for at least 1 year.

Elution buffer

To make 50mL of elution buffer, combine 49.4mL of molecular water, 500µL of 1 M Tris-HCl (pH 8.0), 100µL of 0.5 M EDTA (pH 8.0) and 25µL of Tween 20.

This is sufficient for 10 96-well extraction plates 50µL each). This buffer can be stored at room temperature for at least 1 year.

Option 1: G-Biosciences Silica magnetic beads (GENO78 6-915)

Option 2: MagAttract Suspension G magnetic beads (Qiagen: 1026901)

Note: MagAttract beads are much cheaper and have very similar recovery for fragments 50bp and above, with minor loss of 35bp fragments. SeraSil beads only recover from 75bp (SeraSil 700) or 100bp (SeraSil 400).

The original Rohland protocol washes the beads before use, but we have found unwashed beads work just as effectively. If washing follow these steps:

1. Fully resuspend the stock suspension of silica beads by vortexing.
2. Transfer 10µL of silica bead suspension per reaction to a 2.0-ml lobind tube, including an excess of 5% (e.g., for a plate take 1mL).
3. Place the tube on a magnet to collect the beads.
4. Pipette off and discard the supernatant.
5. Remove the tube from the magnet, add 500µL of elution buffer and resuspend the beads by vortexing for 8 seconds.
6. Spin the tubes briefly in a microcentrifuge to collect the suspension at the bottom.
7. Place the tube back on the magnet to collect the beads.
8. Pipette off and discard the supernatant.
9. Repeat steps 5 to 8 for a total of two washes.
10. Resuspend the beads in a volume of elution buffer equivalent to the initial volume used (e.g., for a plate add 1mL).

Tissue can be added to the plate/tubes either before or after the addition of lysis buffer, however tissue from dried specimens (e.g. pinned insects) should be added to wells/tubes with lysis buffer to prevent static displacement. If tissue was previously stored in ethanol this should be dried off before lysis.

1. Add 90µL of lysis buffer C to each sample tube / well. Volume can be adjusted to ensure coverage of tissue, however be mindful that 5x volume of binding buffer is required further down the protocol.
2. Incubate overnight at 56°C on a shaking incubator. The longer the incubation the more the tissue / specimen is digested. Shorter incubations are recommended for better voucher recovery with whole body lysis.
3. Pipette lysate into a new tube / deep-well plate. If tissue / voucher is to be recovered then add appropriate volume of 80% ethanol to cover sample in original tube / well.
4. Add 450µL of binding buffer D (= 5x volume of lysate) to lysate in tube / deep-well plate.
5. Add 10µL of silica magnetic bead suspension to lysate and binding buffer.
6. Vortex for 5sec.
7. Rotate (mix) the tubes / plates for 15min at room temperature.
8. Spin briefly in a microcentrifuge to collect the suspension at the bottom and place them on an appropriate magnet.
9. Once solution clears pipette off the supernatant and discard.
10. Remove from the magnet, add 250µL of wash buffer and vortex for 8sec.
11. Spin briefly to collect the suspension at the bottom and place back on the magnet.
12. Repeat wash (steps 9 - 11) twice for a total of three washes.
13. Aspirate any remaining drops of liquid and dry the beads at room temperature by leaving them on the magnet with open lids. Usually a few minutes is sufficient.
14. Remove from the magnet, add 50µL - 100µL of elution buffer depending on original sample size and anticipated DNA recovery.
15. Vortex until all beads have been resuspended and then briefly spin down.
16. Incubate for 2min at room temperature.
17. Place back on the magnet, wait until the solution clears and transfer the supernatant to a fresh tube / plate.

Proceed to library preparation or freeze extracted DNA.

The manual protocol has been tested on the Kingfisher Flex and Apex platforms. The protocol starts after step 5 above, i.e. prepare the deep-well plate with 90µL lysate + 450µL binding buffer D and 10µL silica beads.

The kingfisher protocol uses onboard "tip mixing" rather than vortexing / mixing to resuspend beads and skips all centrifugation steps.