- Materials
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- 50 pg high molecular weight genomic DNA
- REPLI-g® Single Cell Kit (QIAGEN, cat # 150343)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 2 ml Eppendorf DNA LoBind tubes
- 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™ cat # A63881)
- Freshly prepared 80% ethanol in nuclease-free water
- TE buffer: 10 mM Tris (pH 8.0), 0.1 mM EDTA
- PEG 8000, 50% w/v (Rigaku Reagents, cat # 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, cat # 15893661)
- Equipment
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- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Thermal cycler
- Hula mixer (gentle rotator mixer)
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
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Thaw the REPLI-g sc DNA Polymerase on ice, mix well by pipetting and spin down. Store on ice until ready to use.
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Prepare the DNA in nuclease-free water.
- Transfer 50 pg genomic DNA into a clean 0.2 ml thin-walled PCR tube.
- Adjust the volume to 4 μl with nuclease-free water.
- Mix thoroughly by inversion and gently flicking to avoiding unwanted shearing.
- Spin down briefly in a microfuge.
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Reconstitute the Buffer DLB from the QIAGEN REPLI-g Single Cell kit as follows:
- Add 500 µl of nuclease-free water to the Buffer DLB tube.
- Thoroughly mix by vortexing and briefly spin down.
Note: According to the manufacturers, the reconstituted Buffer DLB can be stored for up to 6 months at -20°C.
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In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare sufficient Buffer D2 for the total number of reactions required as follows:
Reagent Volume for 4 samples Volume for 12 samples Volume for 24 samples DTT, 1M 1 µl 3 µl 6 µl Reconstituted Buffer DLB 11 µl 33 µl 66 µl Total 12 µl 36 µl 72 µl Note: The REPLI-g® Mini/Midi Handbook recommends preparing a stock of Buffer D2 minimise risk of error when pipetting small volumes. According to the manufacturers, the prepared Buffer D2 can be stored for up to 3 months at -20°C.
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Add 3 µl of prepared Buffer D2 to the gDNA input sample in the 0.2 ml thin-walled PCR tube.
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Mix gently by flicking the tube and spin down.
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Incubate the reaction at 65°C for 10 minutes.
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Add 3 µl of of Stop Solution to the denatured DNA sample tube. Mix by flicking the tube, briefly spin down and place on ice.
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In a clean 1.5 ml Eppendorf DNA LoBind tube placed on ice, prepare the master mix as follows:
Pipette mix 10-20 times between each addition.
Reagent Volume Nuclease-free water 9 µl REPLI-g sc Reaction Buffer 29 µl REPLI-g sc DNA Polymerase 2 µl Total 40 µl -
Mix thoroughly by pipetting and briefly spin down before storing the master mix on ice.
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Combine the following reagents in the same 0.2 ml thin-walled PCR tube containing the sample:
Reagent Volume Denatured DNA sample (from previous step) 10 µl Prepared master mix 40 µl Total 50 µl -
Mix gently by flicking the tube and spin down.
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Incubate the reaction for 2 hours at 30°C and 3 minutes at 65°C using a thermal cycler.
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Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
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Resuspend the AMPure XP beads by vortexing.
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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 80% 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 80% 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 the eluted sample using a Qubit fluorometer.
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Prepare your amplified DNA sample as follows:
- Transfer 1.5 µg of amplified DNA into a clean 0.2 ml thin-walled PCR tube.
- Adjust the volume to 24 μl with nuclease-free water.
- Mix thoroughly by inversion and gently flicking to avoiding unwanted shearing.
- Spin down briefly in a microfuge.
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Prepare the following reaction in the 0.2 ml thin-walled PCR tube containing the sample by adding the reagents in the following order:
Reagent Volume 1.5 µg of amplified DNA (from previous step) 24 µl NEBuffer 2 3 µl T7 Endonuclease I 3 µl Total 30 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
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Incubate the reaction for 60 minutes at 37°C.
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Prepare the Custom buffer with beads as follows:
- Prepare the Custom buffer mix 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 - Mix the Custom buffer thoroughly by pipetting.
- Resuspend the AMPure XP beads by vortexing.
- Prepare two 1.5 ml Eppendorf DNA LoBind tubes to contain 1 ml of resuspended AMPure XP beads each.
- Pellet the beads in both tubes on a magnet. Keeping the tubes on the magnet, pipette off the supernatants.
- Remove both tubes from the magnet and resuspend the beads in each tube with 1 ml of nuclease-free water. Return the tubes to the magnet and allow the beads to pellet. Pipette off the water and discard.
- Repeat the previous step.
- Spin down and place the tubes back on the magnet. Pipette off any residual water.
- Resuspend both pellets in 200 ul of Custom Buffer. Transfer the full volume of both tubes of resuspended beads to the remaining Custom buffer in the 2 ml Eppendorf LoBind DNA tube.
- Mix the Custom buffer with beads thoroughly by pipetting.
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Prepare the amplified DNA sample as follows:
- Transfer the 30 µl of amplified DNA sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
- Adjust the volume to 50 μl with TE buffer, pH 8.
- Mix thoroughly by inversion and gently flicking to avoiding unwanted shearing.
- Spin down briefly in a microfuge.
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Add 35 µl of the Custom buffer with beads to the DNA sample, and mix by flicking the tube.
Note: Thoroughly mix the Custom buffer by pipetting prior to use to ensure the beads are fully resuspended.
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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 a slightly higher DNA recovery yield is desired.
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Prepare 500 μl of 80% 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 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 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.