- Materials
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- Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
- Consumables
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- Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Equipment
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- Thermal cycler
- Vortex mixer
- Magnetic rack
- Multichannel pipette and tips
- Plate mixer
- Microfuge
- Ice bucket with ice
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The hybrid capture protocol uses the SureSelect Target Enrichment Box 1 reagents (stored at room temperature), as well as Dynabeads MyOne Streptavidin T1 magnetic beads.
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Warm the SureSelect Wash Buffer 2 at 65°C.
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Resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads by vortexing.
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Add 50 µl of the beads to a fresh 1.5 ml Eppendorf DNA LoBind tube.
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Add 200 µl of SureSelect Binding Buffer to the beads.
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Mix by pipetting.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Repeat steps 4–6 twice more for a total of three washes.
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Resuspend the beads in 200 µl of SureSelect Binding Buffer.
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Keep the hybridisation tube at 65°C. Using a multichannel pipette, transfer the whole volume (~25–29 µl) of the hybridisation mixture from the 65°C reaction to the tube containing 200 µl of washed streptavidin beads.
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Pipette up and down until beads are fully resuspended.
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Incubate the tube on a Hula mixer for 30 mins at room temperature. Make sure the sample is mixing in the tube.
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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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Resuspend the beads in 200 µl of SureSelect Wash Buffer 1.
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Pipette up and down until beads are fully resuspended.
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Incubate the reaction for 15 minutes at room temperature.
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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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Resuspend the beads in 200 µl of Wash Buffer 2 pre-warmed at 65°C.
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Pipette up and down until beads are fully resuspended.
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Transfer the sample to a 0.2 ml thin-walled PCR tube.
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Incubate the tube for 10 minutes at 65°C in the thermal cycler.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
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Repeat the wash steps 17–22 twice more for a total of three washes. Make sure all of the wash buffer has been removed during the final wash.
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Add 96 µl of nuclease-free water to the sample, and pipette up and down to resuspend the beads. Keep the sample on ice.
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Captured DNA remains on the streptavidin beads during the post-capture amplification step.