- Materials
-
- 6 million PBMCs isolated from whole blood
- Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
- Consumables
-
- Monarch® HMW DNA Extraction Kit for Tissue (NEB, T3060)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- Ethanol, 100% (e.g. Fisher, 16606002)
- 5 ml Eppendorf DNA LoBind tubes
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
-
- Thermal cycler
- Temperature-controlled centrifuge
- Microfuge
- Hula mixer (gentle rotator mixer)
- Vortex mixer
- Qubit fluorometer (or equivalent)
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Wide-bore pipette tips
- Ice bucket with ice
-
Day 2: Ultra-long DNA experiment overview
During the first day of the Ultra-long DNA experiment, the ultra-high molecular weight (uHMW) gDNA is extracted using the NEB Monarch HMW DNA Extraction Kit for Tissue. An optional uHMW gDNA quantification step has also been included. However, this step can be omitted and 750 µl of DNA in Extraction EB (EEB) can be taken straight into step 39 for tagmentation. During tagmentation, the DNA is cleaved and the transposase adapter simultaneously added to prepare the DNA ends for the attachment of the rapid adapter. The DNA is then cleaned using Precipitation Buffer (PTB) and incubated overnight.
-
Thaw the Extraction EB (EEB) at room temperature, mix by vortexing and place on ice.
-
Transfer 6 million cells resuspended in 40 µl PBS to a fresh 5 ml tube.
-
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.
-
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.
Note: the first bead is sacrificial and will remain stuck 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.
-
Optional actionAt this point, the sample can be stored overnight at room temperature.
The next steps for DNA quantification are optional. Continue to the next stage of the protocol if quantification is to be omitted.
-
Use a regular P200 pipette tip to aspirate 10 µl of gDNA.
-
Dispense the aspirated gDNA into a fresh 2 ml Eppendorf DNA LoBind tube.
-
Add a Monarch DNA Capture Bead to the 10 µl of gDNA and vortex aggressively for 1 minute to shear the gDNA.
-
Transfer the gDNA and beads into a clean Monarch Bead Retainer inserted in a Monarch Collection Tube II. Spin the tube at 1000 x g for 1 minute to separate gDNA from the beads. Dispose of beads and bead retainer.
-
Transfer the gDNA into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify the sample using a Qubit fluorometer. The expected yield is 30-40 µg of DNA.
-
Thaw the the kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:
Once thawed, keep all the kit components on ice.
Reagent Thaw at room temperature Briefly spin down Mix well by pipetting Fragmentation Mix (FRA) Not frozen ✓ ✓ FRA dilution buffer (FDB) Not frozen ✓ ✓ Rapid Adapter (RA) Not frozen ✓ ✓ -
In a 1.5 ml Eppendorf DNA LoBind tube, dilute the Fragmentation Mix (FRA) with FRA Dilution Buffer (FDB) as follows:
Reagent Volume Fragmentation Mix (FRA) 6 µl FRA dilution buffer (FDB) 244 µl Total 250 µl -
Mix the diluted Fragmentation Mix (FRA) by pipetting.
-
Using a regular pipette tip, add 250 µl of diluted Fragmentation Mix (FRA) to the 750 µl of extracted DNA. Stir the reaction with the pipette tip whilst expelling the diluted Fragmentation Mix (FRA) to ensure an even distribution.
-
Immediately mix the reaction by slowly pipetting 10 times with a wide-bore pipette tip.
Visually check the reagents are thoroughly mixed. It is important to immediately mix the diluted Fragmentation Mix (FRA) with the DNA thoroughly.
-
Incubate the reaction as follows:
Temperature Time Room temperature 10 minutes 75°C 10 minutes On ice Cool on ice for a minimum of 10 minutes Note: the reaction must be cooled on ice before adding Rapid Adapter (RA) to prevent denaturing the enzyme.
-
Add 5 µl Rapid Adapter (RA) to the reaction using a regular pipette tip.
-
Gently mix the reaction by slowly pipetting five times using a 1 ml wide-bore pipette tip.
Note: visually check to ensure the reaction is thoroughly mixed.
-
Incubate the reaction for 30 minutes at room temperature.
-
Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by vortexing as indicated by the table below:
Reagent Thaw at room temperature Briefly spin down Mix well by pipetting Precipitation buffer (PTB) ✓ ✓ ✓ Elution Buffer (EB) ✓ ✓ ✓ Once thawed, keep all the kit components on ice.
-
Using a regular pipette tip, add 500 µl of Precipitation Buffer (PTB) to the sample.
-
Mix the sample by rotating on a Hula Mixer (rotator mixer) for 20 minutes at 3 rpm.
Visually inspect to check the DNA has precipitated, forming a glassy white mass.
-
Centrifuge the sample at 1000 x g for 1 minute.
-
Using a regular pipette tip, carefully remove the supernatant from the tube, taking care not to aspirate the DNA pellet.
-
Centrifuge the sample at 1000 x g for 1 minute.
-
Using a regular pipette tip, carefully remove any residual supernatant from the tube, taking care not to aspirate the DNA pellet.
-
Using a regular pipette tip, add 300 µl of Elution Buffer (EB) to the tube containing the DNA. Incubate overnight at room temperature, for a minimum of 12 hours.