- Materials
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- Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
- Consumables
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- Cot-1 DNA (ThermoFisher Scientific 15279-011)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 0.2 ml 96-well PCR plate
- Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'
- Equipment
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- SpeedVac
- Thermal cycler
- Ice bucket with ice
- Vortex mixer
- Microfuge
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In a clean 1.5 ml Eppendorf DNA LoBind tube, mix the following:
Reagent Volume DNA library 700-1000 ng Cot-1 DNA 5 µg Blocking oligo top 1 µl -
The volume of the reaction can be variable, as the water is evaporated in step 2. After this, the DNA is reconstituted to a set volume.
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Evaporate the water in a SpeedVac at 45ºC for approximately 1 hour.
Poke one or more holes in the lid with a narrow gauge needle. You can also break off the cap, cover with parafilm, and poke a hole in the parafilm.
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Reconstitute with nuclease-free water to a final volume of 9 µl. Pipette up and down along the sides of the tube for optimal recovery.
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Mix thoroughly by vortexing and spin down for 1 minute.
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Move the 9 µl gDNA library samples to separate wells of a 96-well plate or strip tube. Seal the wells or close the tubes and incubate in the thermal cycler using the following program:
Stage Temperature Time Step 1 95 ºC 5 min Step 2 65 ºC 5 min Step 3 65 ºC Hold -
You will now need to prepare the Hybridisation Buffer mix and the RNase Block ready to be combined with the Capture Library reagent from the SureSelect kit. This will then be combined with the adapted, amplified DNA sample.
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Once the sample tubes are in the thermal cycler, mix the reagents in the table below to make the Hybridisation Buffer:
Reagent Volume for 1 reaction SureSelect Hyb 1 (orange cap or bottle) 6.63 µl SureSelect Hyb 2 (red cap) 0.27 µl SureSelect Hyb 3 (yellow cap or bottle) 2.65 µl SureSelect Hyb 4 (black cap or bottle) 3.45 µl Total 13 µl In the event of precipitation, warm the Hybridisation Buffer at 65°C for 5 minutes.
Otherwise, keep buffer at room temperature until it is used for the Hybridisation mix. -
Dilute the SureSelect RNase Block (purple cap) in nuclease-free water, sufficient for the number of hybridisation reactions in the run. Keep the mixture on ice.
Capture library size RNase block dilution (parts RNase block:water) Volume of diluted RNase block per hybridisation reaction ≥3 Mb 25% (1:3) 2 μl <3 Mb 10% (1:9) 5 μl -
Prepare the Capture Library Hybridisation Mix according to the table below. Only keep the mixture at room temperature until it is added to sample wells.
Reagent Volume for 1 reaction Hybridisation buffer mixture 13 µl 25% RNase Block solution 2 µl Capture library (red cap) ≥3 Mb 5 µl Total 20 µl -
If your capture library is <3 Mb prepare the Capture Library Hybridisation Mix as follows:
Reagent Volume for 1 reaction Hybridisation buffer mixture 13 µl 10% RNase Block solution 5 µl Capture library (red cap) <3 Mb 2 µl Total 20 µl -
Keeping all reagents at 65°C, add 20 µl of the Capture Hybridisation Mix to each well/tube containing 9 µl adapted and amplified DNA sample.
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Mix by pipetting.
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Seal all the wells or replace the caps on the strip tubes.
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Incubate the plate or tubes for 16 to 24 hours at 65 °C with a heated lid set at 105 °C.