- Materials
-
- Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
- Consumables
-
- Cot-1 DNA (ThermoFisher Scientific 15279-011)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 0.2 ml thin-walled PCR tubes
- Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'
- Equipment
-
- SpeedVac
- Thermal cycler
- Ice bucket with ice
- Vortex mixer
- Microfuge
-
In a clean 1.5 ml Eppendorf DNA LoBind tube, mix the following:
Reagent Volume DNA library 300–1000 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.
-
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.
-
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.
-
Mix thoroughly by vortexing and spin down for 1 minute.
-
Move the 9 µl DNA library sample to a 0.2 µl thin-walled PCR tube, close the tube and incubate in the thermal cycler using the following program:
Stage Temperature Time Step 1 95°C 5 min Step 2 65°C 5 min Step 3 65°C 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.
-
Once the sample tube is 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. Keep the mixture on ice.
RNase block dilution (parts RNase block:water) Volume of diluted RNase block 25% (1:3) 2 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 tube.
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 -
Keeping all reagents at 65°C, add 20 µl of the Capture Hybridisation Mix to the tube containing 9 µl adapted and amplified DNA sample.
-
Mix by pipetting.
-
Replace the cap on the tube.
-
Incubate the tube for 16–24 hours at 65°C with a heated lid set at 105°C.