- Materials
-
- Ligation Adapter (LA)
- Ligation Buffer (LNB)
- Short Fragment Buffer (SFB)
- AMPure XP Beads (AXP)
- Elution Buffer (EB)
- Consumables
-
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
-
- Magnetic rack
- Hula mixer (rotator mixer)
- Microfuge
- Vortex mixer
- P1000 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
-
Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.
-
Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
-
Thaw the Elution Buffer (EB) at room temperature and mix by vortexing. Then spin down and place on ice.
-
Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.
-
In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
Between each addition, pipette mix 10-20 times.
Reagent Volume DNA sample from the previous step 60 µl Ligation Buffer (LNB) 25 µl NEBNext Quick T4 DNA Ligase 10 µl Ligation Adapter (LA) 5 µl Total 100 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
-
Incubate the reaction for 10 minutes at room temperature.
-
Resuspend the AMPure XP Beads (AXP) by vortexing.
-
Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
-
Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
-
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 250 μl of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Note: Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.
-
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 the pellet in 34 µl Elution Buffer (EB).
-
Spin the sample down briefly and incubate for 10 minutes at room temperature.
Note: For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.
-
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
-
Remove and retain 34 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
-
Quantify 1 µl of eluted sample using a Qubit fluorometer.
-
Prepare 50–100 fmol of your final library to 32 µl with Elution Buffer (EB).
Alternatively, assume an average fragment length of 1 kbp and proceed with 33 ng of library.
Note: If your DNA library is below the required concentration, take forward the full volume of 32 µl eluted DNA for sequencing.
Caution: Please note, very low recovery could be indicative of library preparation failiure.