- Materials
-
- Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
- Consumables
-
- Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
- 0.2 ml 96 well PCR plate
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Equipment
-
- Thermal cycler
- Vortex mixer
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Multichannel pipette and tips
- Plate mixer
- Centrifuge capable of taking 96-well plates
- Ice bucket with ice
-
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.
-
Warm the SureSelect Wash Buffer 2 at 65°C.
-
Resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads by vortexing.
-
Add 50 µl of the beads to wells of a fresh PCR plate or strip tube, one well for each hybridisation sample.
-
Add 200 µl of SureSelect Binding Buffer to the beads.
-
Mix by pipetting.
-
Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
-
Repeat steps 4–6 twice more for a total of three washes.
-
Resuspend the beads in 200 µl of SureSelect Binding Buffer.
-
Keep the hybridisation plate or strip tube at 65 °C. Using a multichannel pipette, transfer the whole volume (approximately 25 to 29 µl) of each hybridisation mixture from the 65 °C reaction to the plate or strip tube wells containing 200 µl of washed streptavidin beads.
-
Pipette up and down until beads are fully resuspended.
-
Seal all the wells or replace the caps on the strip tubes.
-
Incubate the plate or strip tube on a 96-well plate mixer, mixing vigorously (1400–1800 rpm) for 30 minutes at room temperature. Make sure the samples are mixing in the wells.
-
Spin down the samples in a centrifuge or mini-plate spinner.
-
Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
-
Resuspend the beads in 200 µl of SureSelect Wash Buffer 1.
-
Pipette up and down until beads are fully resuspended.
-
Incubate the reaction for 15 minutes at room temperature.
-
Spin down the samples in a centrifuge or mini-plate spinner.
-
Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
-
Resuspend the beads in 200 µl of Wash Buffer 2 pre-warmed at 65°C.
-
Pipette up and down until beads are fully resuspended.
-
Seal all the wells or replace the caps on the strip tubes.
-
Incubate the plate or strip tube for 10 minutes at 65 °C on the thermal cycler.
-
Place on a magnetic rack, allow beads to pellet and pipette off supernatant.
-
Repeat the wash steps 20 - 24 twice more for a total of three washes. Make sure all of the wash buffer has been removed during the final wash.
-
Add 96 µl of nuclease-free water to each sample well, and pipette up and down to resuspend the beads. Keep the samples on ice.
-
Captured DNA remains on the streptavidin beads during the post-capture amplification step.