- Materials
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- 5 x 10^6 cells
- Consumables
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- Puregene Cell Kit (QIAGEN, 158043)
- Freshly prepared 70% ethanol in nuclease-free water
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- 1 x Phosphate-buffered saline (PBS)
- Isopropanol
- Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 15 ml Falcon tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Incubator or water bath set at 37°C and 50°C
- Vortex mixer
- Inoculation loop or disposable tweezers for spooling DNA
- Wide-bore pipette tips
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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Extraction from cultured cell lines:
Extract DNA from your sample(s) using one of our recommended extraction protocols.
For the benchmarking of this method, the Oxford Nanopore team extracted DNA from ~5 million cells using the protocol: Human cell line DNA – QIAGEN Puregene Cell Kit. The steps for this method are outlined below.
Note: this method is also suitable for mouse cell line DNA.We also offer multiple mammalian sample extraction protocols, which you can use for other sample types.
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Harvest and pellet 5 x 10^6 cells by centrifugation at 300 x g for 3 minutes. If any liquid remains associated with the pellet, spin down the cells again and aspirate the remaining supernatant.
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Add 200 µl of 1x PBS to the pelleted cells and centrifuge at 300 x g for 3 minutes. Aspirate and discard the supernatant.
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Add 2 ml of Cell Lysis Solution to the washed cell pellet. Using a wide-bore pipette tip, resuspend the cells and transfer them to a 15 ml Falcon tube. If clumps of cells remain, gently invert the tube.
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Incubate the sample at 37°C for 30 minutes.
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Add 700 µl of the Protein Precipitation Solution to the lysed cells and mix by vortexing for three pulses of 5 seconds.
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Centrifuge the sample at 2000 x g for 5 minutes.
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Transfer the supernatant to a new tube and add 2.5 ml of room temperature isopropanol. Discard the pellet.
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Mix by gently inverting the tube 50 times.
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Spool the DNA using an inoculation loop or disposable tweezers.
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Dip the spooled DNA in an Eppendorf tube containing 70% cold ethanol.
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Remove the inoculation loop or tweezers with the spooled DNA from the ethanol tube, and allow it to air-dry for a few seconds.
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Dip the DNA in a 1.5 ml Eppendorf DNA LoBind tube containing 250 µl TE (1 mM EDTA, pH 8.0) and allow the DNA to gently dislodge from the loop/tweezers.
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Incubate the DNA pellet for 2 hours at 50°C, occasionally mixing the tube contents by gentle inversion.
Note: The pellet may take some time to dissolve, so ensure the solution is homogenous before quantifying.
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Quantify 1 µl of each eluted sample using a Qubit fluorometer.