- Materials
-
- Long Fragment Buffer (LFB)
- Short Fragment Buffer (SFB)
- Elution Buffer (EB)
- SPRI Dilution Buffer (SDB)
- Consumables
-
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
-
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Eppendorf 5424 centrifuge (or equivalent)
-
This step removes excess unligated adapters and other short DNA fragments, and concentrates and buffer-exchanges the library in preparation for sequencing.
-
Thaw the Elution Buffer (EB) and SPRI Dilution Buffer (SDB) at room temperature, mix by vortexing, spin down and place on ice.
-
To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.
-
To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
-
Add 1 volume (80 µl) of the SPRI Dilution Buffer (SDB) to the ligation mix. Mix gently by flicking the tube.
-
Resuspend the AMPure XP beads by vortexing.
-
Add 0.3x volume (48 µl) of AMPure XP Beads to the ligation sample. The volume of beads is calculated based on the volume after the addition of SDB. Mix gently by inversion. If any sample ends up in the lid, spin down the tube very gently, keeping the beads suspended in liquid.
-
Incubate the sample for 10 minutes at room temperature. Do not agitate or pipette the sample.
-
Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.
-
Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB), depending on the size of your target molecule. Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
-
Repeat the previous step.
-
Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
-
Remove the tube from the magnetic rack and resuspend pellet in 13 µl Elution Buffer (EB). Incubate for 10 minutes at room temperature.
Note: For targets >30 kb, we recommend increasing the elution time to 30 minutes.
-
Pellet the beads on a magnet until the eluate is clear and colourless.
-
Remove and retain 12 µl of eluate which contains the DNA library in a clean 1.5 ml Eppendorf DNA LoBind tube.
- Dispose of the pelleted beads
-
Optional actionIf quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.
Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX001), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer (SQB) and Loading Beads (LB), required for loading the libraries onto flow cells.