- Materials
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- 3 ml of whole blood
- Custom SPRI bead suspension
- Consumables
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- Puregene Blood Kit (QIAGEN, 158023)
- Freshly prepared 80% ethanol in nuclease-free water
- Isopropanol, 100% (Fisher, 10723124)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- 1.5 ml Eppendorf DNA LoBind tubes
- 15 ml Falcon tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Equipment
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- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Heating block
- Vortex mixer
- Qubit fluorometer (or equivalent for QC check)
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Whole blood gDNA extraction for the Duplex experiment
An input of 1 µg of gDNA must be prepared for the Duplex experiment. You may extract the gDNA by any means is most appropriate. This section outlines how to extract gDNA from whole blood using the QIAGEN Puregene Blood Kit.
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Perform cell separation and lysis according to the QIAGEN Puregene Handbook for 3 ml of blood (pages 19–20, steps 1–7):
Dispense 9 ml of RBC Lysis Solution into a 15 ml Falcon tube.
Add 3 ml of whole blood and mix by inverting 10 times.
Incubate for 5 minutes at room temperature. Invert at least once during the incubation.
Centrifuge for 2 minutes at 2000 x g to pellet the white blood cells.
Carefully discard the supernatant by pipetting or pouring, leaving approximately 200 µl of the residual liquid and the white blood cell pellet.
Vortex the tube vigorously to resuspend the pellet in the residual liquid. Vortexing greatly facilitates cell lysis in the next step.
Add 3 ml of Cell Lysis Solution and pipette mix to lyse the cells or vortex for 10 seconds.
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Incubate the samples at 37°C for 30 minutes. If the sample is not homogenous, gently invert the tubes and extend the incubation for another 30 minutes.
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Purify the lysate according to the QIAGEN Puregene Handbook for 3 ml of blood (pages 20–21, steps 8–17):
Add 15 µl of RNase A Solution and mix by inverting 25 times. Incubate for 15 minutes at 37°C. Then incubate for 3 minutes on ice to quickly cool the sample.
Add 1 ml of Protein Precipitation Solution and vortex vigorously for 20 seconds at high speed.
Centrifuge for 5 minutes at 2000 x g. The precipitated proteins should form a tight brown pellet. If the protein pellet is not tight, incubate on ice for 5 minutes and repeat the centrifugation.
Pipette 3 ml of isopropanol into a clean 15 ml Falcon tube and add the supernatant from the previous step by pouring carefully. Be sure that the protein pellet is not dislodged during pouring.
Mix by inverting 50 times until the DNA is visible as threads or a clump.
Centrifuge for 3 minutes at 2000 x g. The DNA may be visible as a small white pellet.
Carefully discard the supernatant and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube.
Add 3 ml of 80% ethanol and invert several times to wash the DNA pellet.
Centrifuge for 1 minute at 2000 x g.
Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper, taking care that the pellet remains in the tube. Dry the pellet for 5-10 minutes. The pellet might be loose and easily dislodged. Avoid over-drying the DNA pellet as the DNA will be difficult to dissolve.
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To maximize the DNA yield, we recommend that the elution is performed for 2 hours at 50°C, using 150 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), occasionally mixing the tube contents by gentle inversion.
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Quantify 1 µl of eluted sample using a Qubit fluorometer.
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Dilute your DNA sample to 60 ng/µl in a final volume of 50 µl of TE buffer at pH 8.
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Add 0.7X (35 µl) of room temperature custom SPRI bead suspension to your DNA sample, and mix by flicking the tube.
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Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.
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Spin down briefly and pellet on a magnet until the 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 80% 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 magnetic rack. Pipette off any residual ethanol. Allow the pellet 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 40 µl of TE buffer. Incubate for 1 minute at 50°C, and then for 5 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
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Remove and retain 40 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of size-selected DNA using a Qubit fluorometer.
You can expect a 50-55% loss of DNA depending on a fragment length distribution of input material: the greater the proportion of short fragments (<1.5-2 kb), the greater the sample loss.