- Materials
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- 750 µl of extracted uHMW gDNA in EEB
- Rapid Adapter (RA)
- Fragmentation Mix (FRA)
- FRA Dilution Buffer (FDB)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Thermal cycler or heat block
- Microfuge
- Wide-bore pipette tips
- P1000 pipette and tips
- P20 pipette and tips
- Ice bucket with ice
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Best practice for handling uHMW gDNA
When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.
To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.
While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.
For further information, please refer to the troubleshooting section.
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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.
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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.
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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.
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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.
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Add 5 µl Rapid Adapter (RA) to the reaction using a regular pipette tip.
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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.
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Incubate the reaction for 30 minutes at room temperature.