TITLE: MinION DNA Library Prep (SQK-LSK109)
AUTHORS: Victoria Jackson, Michelle Michelsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for ligation sequencing DNA library prep for MinION slightly modified from the ONT protocol.</div></div>

[STEPS]
?. [Bead clean-up 1]
Make up  of DNA sample in nuclease-free water to a volume of  in an Eppendorf DNA LoBind tube.Make  of fresh 70% ethanol. Place an 1.5 mL tube of nuclease-free water on a heat block set to .
1 µg
49 µl
150 µl
55 °C
?. [Bead clean-up 1]
Resuspend the by vortexing.Add  to the DNA and gently flick the tube for  .
[AMPure beads]
?. [Bead clean-up 1]
Place the tube on the magnetic rack and wait for the beads to pellet on the magnet. Once the beads have pelleted, pipette off and discard the supernatant.
?. [Bead clean-up 1]
Without disturbing the pellet on the magnet, wash the beads with . Pipette off the ethanol and discard.
[70% ethanol]
?. [Bead clean-up 1]
Allow the bead pellet to dry on the magnet with the tube's lid open for about  , but do not allow the pellet to dry out completely.
?. [Bead clean-up 1]
Remove the tube from the magnet. Elute the DNA with  at  . Gently flick the tube for   to resuspend the beads and replace the tube on the magnet. Once the beads have pelleted, pipette off and retain  of the eluate. Discard the beads.
[nuclease-free water]
55 °C
50 µl
?. [Qubit 1]
Follow the Qubit DNA HS Assay protocol to quantify  of clean DNA from the clean-up step.
1 µl
?. [DNA repair & end-prep]
Thaw DNA CS at  , mix by pipetting and place  .Thaw and .
0 Room temperature
on ice
on ice
?. [DNA repair & end-prep]
In a 0.2 mL PCR tube, mix:
[DNA CS]
[DNA sample]
[NEBNext FFPE DNA Repair Buffer]
[NEBNext FFPE DNA Repair Mix]
[Ultra II End-prep Reaction Buffer]
[Ultra II End-prep Enzyme Mix]
?. [DNA repair & end-prep]
Mix by gently flicking the tube, then spin down.Incubate at  for  , then at  for  using a thermocycler.
20 °C
65 °C
?. [Bead clean-up 2]
Resuspend the by vortexing.Transfer the DNA mixture from the PCR tube to an Eppendorf DNA LoBind tube.
?. [Bead clean-up 2]
Add  to the tube and flick the tube to mix.
[AMPure beads]
?. [Bead clean-up 2]
Incubate on a rotator mixer or invert the tube by hand for  at  .Meanwhile, prepare  using nuclease-free water.
0 Room temperature
[70% ethanol]
?. [Bead clean-up 2]
After the incubation, spin down the sample using a microfuge and place on the magnetic rack.Once a pellet has formed, pipette off the supernatant, leaving tube on the magnet.
?. [Bead clean-up 2]
With the tube still on the magnet, wash the beads without disturbing the pellet with . Pipette off the ethanol and discard.
[70% ethanol]
?. [Bead clean-up 2]
Spin down the tube using a microfuge and replace on the magnet - pipette off residual ethanol and leave the lid open to allow the pellet to dry for around  .
?. [Bead clean-up 2]
Remove the tube from the magnet and resuspend the pellet in  heated to  .Incubate for  at  .
[nuclease-free water]
55 °C
0 Room temperature
?. [Bead clean-up 2]
Replace the tube on the magnet to pellet the beads.Remove and retain  of the eluate in an Eppendorf DNA LoBind tube.
61 µl
?. [Qubit 2]
Follow the Qubit DNA HS Assay protocol to quantify  of DNA from the previous step.
1 µl
?. [Adapter ligation and clean-up]
Spin down AMX and NEBNext Quick T4 DNA Ligase. Place  .Thaw LNB at  , spin down and mix by pipetting. Place  .Thaw EB at , vortex to mix and place  .Thaw LFB or SFB at  , vortex to mix and place  .
on ice
0 Room temperature
on ice
0 Room temperature
on ice
0 Room temperature
on ice
?. [Adapter ligation and clean-up]
In an Eppendorf DNA LoBind tube, mix:Gently flick the tube to mix, then spin down.Incubate the tube at  for  .
[DNA sample]
[LNB]
[NEBNext Quick T4 DNA Ligase]
[AMX]
0 Room temperature
?. [Bead clean-up 3]
Vortex the AMPure beads to resuspend.Add  to the reaction mix from the previous step. Gently flick the tube to mix.
[AMPure beads]
?. [Bead clean-up 3]
Incubate the tube on a rotator mixer for  at .
0 Room temperature
?. [Bead clean-up 3]
Spin down using a microfuge and place the tube on the magnetic rack.Once a pellet has formed, pipette off and discard the supernatant.
?. [Bead clean-up 3]
Add  or  to the tube. Gently flick the tube to resuspend the beads.Spin the tube down briefly, then replace on the magnetic rack.Once a pellet has formed, pipette off the supernatant and discard.
[LFB]
[SFB]
?. [Bead clean-up 3]
?. [Bead clean-up 3]
Using a microfuge, spin down the tube and replace on the magnet.Pipette off any residual supernatant and discard.
?. [Bead clean-up 3]
Allow the pellet to dry with the lid open for about .
?. [Bead clean-up 3]
Remove the tube from the magnetic rack.Add  and gently flick the tube to resuspend the pellet. Incubate for  at .
[EB]
37 °C
?. [Bead clean-up 3]
Spin down very briefly using a microfuge and replace the tube on the magnet.Once a pellet has formed slowly pipette off and retain  of the eluate and place .
15 µl
on ice
?. [Qubit 3]
Follow the Qubit DNA HS Assay protocol to quantify  of the DNA library from the previous step.
1 µl
?. [Bead clean-up 1]
?. [Bead clean-up 2]
?. [Priming the flow cell]
Thaw SQB, LB, FLT and a fresh tube of FB at . Once thawed, place FB .Mix SQB, FB and FLT by vortexing, spin down using a microfuge and place .
0 Room temperature
on ice
on ice
?. [Priming the flow cell]
Open the lid of the MinION. Slide the MinION SpotON flow cell under the MinION clip.
?. [Priming the flow cell]
Rotate open the priming port cover.Place a P1000 pipette tip (pipette set to 200 μL) upright in the priming port. Slowly turn the volume dial to ~220 μL. There should be a small volume of yellow buffer in the pipette tip.Still holding the pipette in an upright position, swiftly and smoothly lift the pipette out of the priming port.
?. [Priming the flow cell]
Add  directly to a fresh tube of FB to make the flow cell priming mix.Vortex to mix.
[FLT]
?. [Priming the flow cell]
Load  of the priming mix into the priming port:Hold the pipette upright in the priming port and turn the dial to gradually to force the priming mix into the flow cell. (To avoid the introduction of air, you may need to turn the dial a small amount in the opposite direction to draw up some of the flow cell buffer and establish liquid-to-liquid contact between the buffer and priming mix.)Wait for  .
800 µl
?. [Preparing the loading library]
Thoroughly mix LB to resuspend the beads by pipetting.
?. [Preparing the loading library]
In an Eppendorf LoBind tube, mix: (freshly mixed)
[SQB]
[LB]
[DNA library]
?. [Loading the flow cell]
Lift the SpotON sample port cover and load an additional  of the priming mix into the still open priming port.
200 µl
?. [Loading the flow cell]
Mix the loading library by slowly pipetting to resuspend the loading beads.
?. [Loading the flow cell]
Slowly, drop  loading library into the SpotON sample port. Make sure the priming port is still open during this step and the library should easily fall into the sample port.
75 µl
?. [Loading the flow cell]
Close the priming port and replace the SpotON sample port cover.Close the lid of the MinION device.Start the sequencing experiment on the MinKNOW software.
?. [Priming the flow cell]
Check the number of pores on the flow cell by navigating to and selecting Flow Cell Check on the MinKNOW software. This should take approximately  .