- Materials
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- 10 pg high molecular weight genomic DNA
- Qiagen REPLI-g Midi Kit
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- T7 Endonuclease I (NEB, cat # M0302)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
- TE buffer: 10 mM Tris (pH 8.0), 0.1 mM EDTA
- PEG 8000, 50% w/v (Rigaku Reagents, 25322-68-3)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 5 M NaCl (Sigma, 71386)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- Equipment
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- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Steps 2-8 are from the REPLI-g protocol, included here for completeness.
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Prepare the DNA in nuclease-free water.
- Transfer 10 pg genomic DNA into a DNA LoBind tube
- Adjust the volume to 5 μl with nuclease-free water
- Mix thoroughly by inversion avoiding unwanted shearing
- Spin down briefly in a microfuge
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Reconstitute the DLB buffer and Stop Solution from the Qiagen REPLI-g Midi kit as follows:
DLB:
Reagent Volume DLB buffer 9 µl Nuclease-free water 32 µl Total 41 µl Stop Solution:
Reagent Volume Stop Solution 12 µl Nuclease-free water 68 µl Total 80 µl -
In a clean 1.5 ml Eppendorf DNA LoBind tube, mix the following:
Reagent Volume Input gDNA, 10 pg 5 µl Reconstituted DLB buffer 5 µl Total 10 µl -
Incubate the reaction for 3 minutes at room temperature.
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Add 10 µl of reconstituted Stop Buffer to the reaction and mix by pipetting.
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In a clean 1.5 ml Eppendorf DNA LoBind tube, mix the following:
Reagent Volume REPLI-g Midi Reaction Buffer 29 µl REPLI-g Midi DNA Polymerase 1 µl Total 30 µl -
Add the REPLI-g Midi polymerase mastermix to the DNA reaction, and mix by pipetting.
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Transfer the sample to a clean 0.2 ml PCR tube, and incubate for 16 hours at 30° C and 3 minutes at 65° C using the thermal cycler.
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Resuspend the AMPure XP beads by vortexing.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Add 90 µl of resuspended AMPure XP beads to the amplification reaction and mix by pipetting.
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Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow 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 pellet in 100 µl nuclease-free water. Incubate for 2 minutes at room temperature.
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Pellet the beads on a magnet until the eluate is clear and colourless.
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Remove and retain 100 µl of eluate in a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of eluted sample using a Qubit fluorometer.
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In a clean 0.2 ml PCR tube, mix the reagents in the following order:
Reagent Volume 1.5 µg of amplified DNA x µl NEBuffer 2 3 µl T7 Endonuclease I 1.5 µl Nuclease-free water 25.5-x µl Total 30 µl -
Incubate the reaction for 15 minutes at 37° C.
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Resuspend the AMPure XP beads by vortexing.
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Prepare the Custom buffer in a clean 2 ml Eppendorf DNA LoBind tube:
Reagent Volume 1 M Tris-HCl 20 μl 0.5 M EDTA pH 8 4 μl 5 M NaCl 640 μl PEG 8000 440 μl Nuclease-free water 888 μl Total 1992 μl -
Transfer thoroughly mixed Agencourt AMPure XP beads into two 1.5 ml Eppendorf DNA LoBind tubes, so that each contains 1 ml.
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Pellet the beads on a magnet. Keeping the tube on the magnet, pipette off the supernatant.
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Wash the beads with 1 ml of nuclease-free water by resuspending the pellet. Return the tube to the magnetic rack, allow beads to pellet, remove the water 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 magnet. Pipette off any residual water.
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Pool the two bead pellets together by resuspending them in 200 µl of Custom buffer. Then transfer the beads into the remaining Custom buffer.
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Make up the amplified DNA sample to a total volume of 50 µl in TE buffer, pH 8.
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Add 35 µl of the custom bead suspension with beads to the 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. This step may be extended to 20 minutes if more efficient ligation is desired.
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Prepare 500 μl of fresh 70% ethanol in nuclease-free water.
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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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Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% 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 magnet. Pipette off any residual supernatant. Allow 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 pellet in 49 µl nuclease-free water. 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.
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Remove and retain 49 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
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Quantify 1 µl of DNA using a Qubit fluorometer - recovery aim ~700 ng.