Nanopore (SQK-LSK109) without barcode

In a 200μl PCR tube, mix the following:

A	B	C
Reagent	Volume	Color
49~100fmol sample DNA + DI Water	50μl
End Repair&A-Tailing Buffer (KAPA)	7μl	Purple
End Repair&A-Tailing Enzyme Mix (KAPA)	3μl	Purple
Total	60μl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Using a thermal cycler, incubate at 20°C for 0h 30m 0s and 65°C for 0h 30m 0s. Hold at 4°C

1.2

Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.

Thaw Ligation Buffer at room temperature, spin down and mix by vortex.

In a 200μl PCR tube, mix in the following order:

A	B	C
Reagent	Volume	Color
End repaired & A-tailing product	60μl
Adapter Mix (AMX)	5μl	Green
DI Water	5μl
Ligation Buffer (KAPA)	30μl	Yellow
NEBNext Quick T4 DNA Ligase (KAPA)	10μl	Yellow
Total	110μl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Using a thermal cycler, incubate at 20°C 0h 30m 0s .

A	B	C
Reagent	Volume	Color
Ligation product	110μl
Agencourt AMPure XP beads	44μl	Stored in 4°C
Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB)	250μl (twice)	Orange & Grey
Elution Buffer from the Oxford Nanopore kit (EB)	14μl	Black

Transfer the DNA sample (110μl) to a clean 1.5 ml tube.

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 14 μl Elution Buffer (EB).

Spin down and incubate for 10 minutes at room temperature.

Remove and retain 14 μl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA tube.

Quantify 1 μl of eluted sample using a Qubit fluorometer.

Resuspend the AMPure XP beads by vortexing.

Add 44 μl (0.4X) of resuspended AMPure XP beads to the adapter-ligated reaction and mix by pipetting.

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.

Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl 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.

Repeat the previous step 3.6.

Thaw the flow cell, Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature.

Open the GridION and slide the flow cell under the clip.

Complete the flow cell priming:

1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

2. Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Mix the prepared library gently by pipetting up and down just prior to loading.

Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION.

Check flow cell (Check the pore)

Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by pipetting and spin down at room temperature.

Remove the flow cell from the machine and slide the priming port clockwise to open the priming port.

After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):

1. Set a P1000 pipette to 200 μl.

2. Insert the tip into the priming port.

3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.

To prepare the flow cell priming mix, add 30 μl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

A	B	C
Reagent	Volume	Color
Flush Tether (FLT)	30μl	Purple
Flush Buffer (FB)	New one	Blue
Total	1.2ml

Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.

Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.

Thoroughly mix the contents of the Loading Beads (LB) by flick.

In a new tube, prepare the library for loading as follows:

A	B	C
Reagent	Volume	Color
Sequencing Buffer (SQB)	37.5μl	Red
Loading Beads (LB),mixed immediately before use	25.5μl	Pink
DNA library	12μl
Total	75μl

Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.

Thaw one tube of Wash Diluent (DIL) at room temperature.

Load 400 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.

Close the priming port and wait for 30 minutes.

Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.

Using a P1000, remove all fluid from the waste channel through Waste port 1. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, spin down briefly and place on ice.

In a clean 1.5 ml Eppendorf DNA tube, prepare the following Flow Cell Wash Mix:

A	B	C
Component	Volume	Color
Wash Mix (WMX)	2μl	Brown
Wash Diluent (DIL)	398μl	Brown
Total	400μl

Mix well by pipetting, and place on ice. Do not vortex the tube.

Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.

Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.

Using a P1000, remove all fluid from the waste channel through Waste port 1.

As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

Rotate the flow cell priming port cover clockwise so that the priming port is visible.

Check for air between the priming port and the sensor array. If necessary, using a P1000 draw back a small volume to remove any air (a few μls):

1. Set a P1000 pipette to 200 μl.

2. Insert the tip into the priming port.

3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer/liquid entering the pipette tip.

4. Visually check that there is continuous buffer from the priming port across the sensor array.

Thaw one tube of Storage Buffer (S) at room temperature.

Mix contents thoroughly by pipetting and spin down briefly.

Rotate the flow cell priming port cover clockwise so that the priming port is visible.

Check for air between the priming port and the sensor array. If necessary, using a P1000 draw back a small volume to remove any air (a few μls):

1. Set a P1000 pipette to 200 μl.

2. Insert the tip into the priming port.

3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer/liquid entering the pipette tip.

4. Visually check that there is continuous buffer from the priming port across the sensor array.

Slowly add 500 μl of Storage Buffer (S) through the priming port of the flow cell.

Close the priming port.

Using a P1000, remove all fluid from the waste channel through Waste port 1. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

The flow cell can now be stored at 4-8°C.

When you wish to reuse the flow cell, remove the flow cell from storage, and allow it to warm to room temperature for ~5 minutes. You will need to perform a Flow Cell Check before loading the next library.