- Materials
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- 5 x 10⁶ cells (e.g. cell culture or tissue sample)
- Consumables
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- Puregene Cell Kit (QIAGEN, 158043)
- Freshly prepared cold 70% ethanol in nuclease-free water
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- 15 ml Falcon tubes
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Inoculation loop or disposable tweezers
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Incubator or water bath set at 37°C and 50°C
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Vortex mixer
- Wide-bore pipette tips
- P1000 pipette and tips
- P200 pipette and tips
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
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Prepare a 1.5 ml Eppendorf DNA LoBind tube with approximately 1 ml of 70% ethanol and store on ice to cool.
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We recommend to harvest and pellet 5 x 10⁶ cells in a 1.5 ml Eppendorf DNA LoBind tube. However, this may vary depending on your sample type. If any liquid remains associated with the pellet, spin down the cells again, then aspirate and discard the remaining supernatant.
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Add 200 µl of 1x PBS to pelleted cells.
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Centrifuge at 300 x g for 3 minutes.
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Without disturbing the pellet, aspirate and discard the supernatant.
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Add 2 ml of Cell Lysis Solution to the washed cell pellet and resuspend the cells using a wide-bore pipette tip.
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Transfer the resuspended cells to a 15 ml Falcon. If clumps of cells remain, gently invert the tube to ensure resuspension.
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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 15 ml falcon tube and add 2.5 ml isopropanol at room temperature. 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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Briefly dip the spooled DNA in the 1.5 ml Eppendorf DNA LoBind tube containing cold 70% ethanol and allow to air dry for a few seconds.
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Transfer the inoculation loop or tweezers with the spooled DNA to a 1.5 ml Eppendorf DNA LoBind tube containing 250 µl TE buffer (10 mM Tris-HCl, 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 by gentle inversion to aid dissolving the pellet. Alternatively, the DNA pellet can be left overnight at room temperature.
Note: The pellet may take some time to dissolve. Ensure the solution is homogenous before proceeding to quantification by gentle inversion and spin down.
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Quantify the sample three times using the Qubit dsDNA BR Assay Kit, ensuring that replicate Qubit measurements are consistent before continuing to the next step.
If the Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended. If this occurs, we recommend increasing the incubation time to aid with resuspension of the DNA pellet.