- Materials
- Consumables
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- Lysing Matrix D, 2 ml tube (MP Biomedical, cat # 116913050-CF)
- HL-SAN nuclease (ArcticZymes Technologies, cat #70910-202)
- MagNA Pure Bacteria Lysis Buffer (Roche, cat # 04659180001)
- Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- TissueLyser LT (QIAGEN, cat # 85600)
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Thermomixer (Eppendorf, Cat# 5382000031)
- Microfuge
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Prepare the HL-SAN nuclease and MagNA Pure Bacteria Lysis Buffer according to the manufacturer's recommendations.
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Transfer 200 µl of the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 10 µl of HL-SAN nuclease to the sample.
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Mix well by pipetting and spin down.
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Incubate at 37°C for 10 minutes at 1000 rpm in a thermomixer.
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Add 200 µl of MagNA Pure Bacterial Lysis Buffer to the reaction.
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Mix well by pipetting and spin down.
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Incubate at room temperature for 10 minutes.
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Resuspend the AMPure XP beads by vortexing.
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Add 400 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Repeat the previous step.
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Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
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Remove the tube from the magnetic rack and resuspend the pellet in 60 µl of nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on the magnet until the eluate is clear and colourless.
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Remove and retain 50 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads.
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Inactivate the sample by heat-treating for 1 hour at 60°C, depending on local health and safety recommendations.