TITLE: Nanopore (SQK-LSK109) without barcode
AUTHORS: Wen-Ting  Zeng

[DESCRIPTION]
Nanopore (SQK-LSK109) without barcode

[STEPS]
SECTION: Step1. DNA repair and end-prep
1.  In a 200μl PCR tube, mix the following:

 
 
 

SECTION: Step1. DNA repair and end-prep
1.1. Ensure the components are thoroughly mixed by pipetting, and spin down.
SECTION: Step1. DNA repair and end-prep
1.2. Using a thermal cycler, incubate at 20 °C for 30 min and 65 °C for 30 min. Hold at 4 °C 
1.2
SECTION: Step2. Adapter ligation
2. Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.

 

SECTION: Step2. Adapter ligation
2.1. Thaw Ligation Buffer at room temperature, spin down and mix by vortex.
 

SECTION: Step2. Adapter ligation
2.2. In a 200μl PCR tube, mix in the following order:

 

SECTION: Step2. Adapter ligation
2.3. Ensure the components are thoroughly mixed by pipetting, and spin down.
SECTION: Step2. Adapter ligation
2.4. Using a thermal cycler, incubate at 20 °C 30 min .
 

SECTION: Step3. Adapter ligation clean-up
3. 
 
 

SECTION: Step3. Adapter ligation clean-up
3.1. Transfer the DNA sample (110μl) to a clean 1.5 ml tube.
SECTION: Step3. Adapter ligation clean-up
3.2. Resuspend the AMPure XP beads by vortexing.
SECTION: Step3. Adapter ligation clean-up
3.3. Add 44 μl (0.4X) of resuspended AMPure XP beads to the adapter-ligated reaction and mix by pipetting. 
SECTION: Step3. Adapter ligation clean-up
3.4. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
SECTION: Step3. Adapter ligation clean-up
3.5. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

 

SECTION: Step3. Adapter ligation clean-up
3.6. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl Short Fragment Buffer (SFB). 

 

SECTION: Step3. Adapter ligation clean-up
3.7. Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. 
 

SECTION: Step3. Adapter ligation clean-up
3.8. Remove the supernatant using a pipette and discard.
SECTION: Step3. Adapter ligation clean-up
3.9. Repeat the previous step 3.6.
SECTION: Step3. Adapter ligation clean-up
3.10. Spin down and place the tube back on the magnet. Pipette off any residual supernatant.
 

SECTION: Step3. Adapter ligation clean-up
3.11. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
SECTION: Step3. Adapter ligation clean-up
3.12. Remove the tube from the magnetic rack and resuspend the pellet in 14 μl Elution Buffer (EB).
SECTION: Step3. Adapter ligation clean-up
3.13. Spin down and incubate for 10 minutes at room temperature.
 

SECTION: Step3. Adapter ligation clean-up
3.14. Remove and retain 14 μl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA tube.
SECTION: Step3. Adapter ligation clean-up
3.15. Quantify 1 μl of eluted sample using a Qubit fluorometer.

 

SECTION: Step4. Priming and loading the SpotON flow cell
4. Thaw the flow cell, Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature.  
SECTION: Step4. Priming and loading the SpotON flow cell
4.1. Open the GridION and slide the flow cell under the clip.
 

  

SECTION: Step4. Priming and loading the SpotON flow cell
4.2. Check flow cell (Check the pore)
 

SECTION: Step4. Priming and loading the SpotON flow cell
4.3. Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by pipetting and spin down at room temperature.

SECTION: Step4. Priming and loading the SpotON flow cell
4.4. Remove the flow cell from the machine and slide the priming port clockwise to open the priming port.
  

SECTION: Step4. Priming and loading the SpotON flow cell
4.5. 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.
 

SECTION: Step4. Priming and loading the SpotON flow cell
4.6. 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.


 
   

SECTION: Step4. Priming and loading the SpotON flow cell
4.7. 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.
 

SECTION: Step4. Priming and loading the SpotON flow cell
4.8. Thoroughly mix the contents of the Loading Beads (LB) by flick.
SECTION: Step4. Priming and loading the SpotON flow cell
4.9. In a new tube, prepare the library for loading as follows:

 
 
SECTION: Step4. Priming and loading the SpotON flow cell
4.10. 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.
 

SECTION: Step4. Priming and loading the SpotON flow cell
4.11. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Step4. Priming and loading the SpotON flow cell
4.12. 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.
SECTION: Step4. Priming and loading the SpotON flow cell
4.13. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION.
   

SECTION: Step5. Flow Cell Wash
5.  
Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
SECTION: Step5. Flow Cell Wash
5.1. Thaw one tube of Wash Diluent (DIL) at room temperature.
SECTION: Step5. Flow Cell Wash
5.2. Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, spin down briefly and place on ice.
SECTION: Step5. Flow Cell Wash
5.3. In a clean 1.5 ml Eppendorf DNA tube, prepare the following Flow Cell Wash Mix:

 
 
SECTION: Step5. Flow Cell Wash
5.4. Mix well by pipetting, and place on ice. Do not vortex the tube.
SECTION: Step5. Flow Cell Wash
5.5. Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
SECTION: Step5. Flow Cell Wash
5.6. Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
SECTION: Step5. Flow Cell Wash
5.7. 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.
   

SECTION: Step5. Flow Cell Wash
5.8. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step5. Flow Cell Wash
5.9. 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.
SECTION: Step5. Flow Cell Wash
5.10. Load 400 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
SECTION: Step5. Flow Cell Wash
5.11. Close the priming port and wait for 30 minutes.
SECTION: Step5. Flow Cell Wash
5.12. Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
SECTION: Step5. Flow Cell Wash
5.13. 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.
 
  

SECTION: Step6. Store the MinION/GridION flow cell for later use
6. Thaw one tube of Storage Buffer (S) at room temperature.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.1. Mix contents thoroughly by pipetting and spin down briefly.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.2. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.3. 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.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.4. Slowly add 500 μl of Storage Buffer (S) through the priming port of the flow cell.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.5. Close the priming port.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.6. 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.

 

  

SECTION: Step6. Store the MinION/GridION flow cell for later use
6.7. The flow cell can now be stored at 4-8°C.
SECTION: Step6. Store the MinION/GridION flow cell for later use
6.8. 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.