- Materials
-
- 6 million cells/nuclei isolated from frozen tissue or white blood cells isolated from whole blood
- Extraction EB (EEB)
- Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
- Consumables
-
- 5 ml Eppendorf DNA LoBind tubes
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- Ethanol, 100% (e.g. Fisher, 16606002)
- 1.5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- Equipment
-
- Heat block set at 56°C
- Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
- Hula mixer (gentle rotator mixer)
- Microfuge
- Wide-bore pipette tips
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Eppendorf 5424 centrifuge (or equivalent)
-
Thaw the Extraction EB (EEB) at room temperature, mix by vortexing and place on ice.
-
Add 6 million cells resuspended in 40 µl PBS to a fresh 5 ml tube. Cells can be isolated from cell culture, white blood cells from blood, or tissue according to the above methods.
-
In a separate 2 ml Eppendorf DNA LoBind tube, combine the following reagents:
Reagent Volume Monarch HMW gDNA Tissue Lysis Buffer 1,800 µl Proteinase K 60 µl Total 1860 µl -
Add 1.8 ml of mixed Monarch HMW gDNA Tissue Lysis Buffer and Proteinase K to the resuspended cells.
-
Gently mix by slowly pipetting the reaction five times using a 1 ml wide-bore pipette tip.
-
Incubate the reaction at 56°C for 10 minutes.
Note: When using cell lines, we have found that this step can be omitted.
-
Using a regular pipette tip, add 15 µl of Monarch RNase A.
-
Gently mix by slowly pipetting the reaction five times using a 1 ml wide-bore pipette tip.
-
Incubate the reaction at 56°C for 10 minutes on a thermomixer at 650 rpm.
-
Using a regular pipette tip, add 900 µl of the Monarch Protein Separation Solution to the reaction and mix using a Hula Mixer (rotator mixer) for 10 minutes, rotating at 3 rpm.
-
Centrifuge the reaction at 16,000 x g for 10 minutes at 4°C to separate the protein from the DNA.
DNA will be present in the upper phase, whereas protein and other contaminants will be in the lower phase.
-
Using a wide-bore pipette tip, carefully aspirate the upper phase containing the DNA and transfer to a fresh 5 ml tube without disturbing the phase below.
The DNA in the upper phase should be extremely viscous and should only be possible to aspirate using a wide-bore pipette tip.
-
Add three Monarch DNA Capture Beads to the collected DNA phase (or to the lysis reaction if proceeded directly from Step 9).
Note: The first bead is a sacrificial bead and will remain at the bottom of the tube throughout the remainder of the process.
-
Add 2.5 ml isopropanol to the tube and mix using a Hula Mixer (rotator mixer) for 20 minutes rotating at 3 rpm. Ensure the DNA has fully precipitated around the glass beads.
-
Leave the tube to stand for 1 minute, without rotating, at room temperature.
-
Aspirate the supernatant from the tube, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any supernatant remaining in the lid of the tube.
Note: if ~100 µl of supernatant is remaining in the tube, perfomance will not be affected.
-
Add 2 ml of Monarch gDNA Wash Buffer to the tube containing DNA bound to the beads and invert the tube to mix.
Ensure ethanol is added to the Monarch gDNA Wash Buffer as per kit guidance.
-
Aspirate the Wash Buffer, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any Wash Buffer remaining in the lid of the tube.
-
Add 2 ml of Monarch gDNA Wash Buffer to the tube containing the DNA bound to the beads.
-
Add 560 µl of Extraction EB (EEB) to a fresh 2 ml Eppendorf tube.
-
Aspirate the Wash Buffer, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any Wash Buffer remaining in the lid of the tube.
-
Transfer the beads to a Monarch Bead Retainer inserted in a Monarch Collection Tube II.
-
Briefly spin the tube using a microfuge to remove any remaining Wash Buffer from the beads. Dispose of the collection tube containing residual wash buffer.
-
Immediately transfer the beads from the bead retainer into the 2 ml tube containing 560 µl of Extraction EB (EEB).
-
Incubate the tube for 10 minutes at 56°C.
-
Pour the eluate and beads into a clean bead retainer inserted in a collection tube. Spin the tube at 1000 x g for 1 minute to separate eluate from the beads. Dispose of beads and bead retainer.
-
Add 200 µl of Extraction EB (EEB) to the collection tube to bring the total elution volume to 760 µl.
-
Transfer the eluate to a fresh 2 ml Eppendorf DNA LoBind tube.
-
Incubate the eluate for 10 minutes at 56°C.
-
Gently mix the eluate by slowly pipetting 10 times using a 1 ml wide-bore pipette tip.
Thorough but gentle resuspension of DNA is required to prevent heterogeneity in the sample.