TITLE: DOH Workshop Protocol Part 3: Library preparation for Rapid Sequencing  DNA V14 Barcoding kit (SQK-RBK114.24) with Pronex modification
AUTHORS: Vesa Qarkaxhija, Bryan Wee, Natalie Ring

[DESCRIPTION]
This protocol performs creation of Nanopore sequencing libraries for the MinION flow cell using the Rapid Barcoding Kit

[BEFORE_START]
Calculate the dilution of your starting genomic DNA from the extraction step to make up 10 µL with a maximum concentration of20 3172- 30 3172 (200 ng-300 ng total DNA).  (For example, if your DNA from the previous protocol was 100 3172, you would only need 2 µLof your sample, diluted in 8 µLof NFW).
Assign each sample to a Barcode (1-24) and note this down.
Program the thermal cycler to incubate at 30 °C for 2 min then 80 °C for another 2 min. Do not start it yet.

[GUIDELINES]
Important throughout the protocol:

 

 


 


 



[STEPS]
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1. Prepare one 0.2 ml thin-walled PCR tube for each sample from the previous step. Label the top of the tube with the barcode number.
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2. In each 0.2 ml thin-walled PCR tubes:
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2.1. Pipette an appropriate amount of your sample (1 µL-10 µL) and add NFW (1 µL-10 µL), if necessary, to get 200 ng-300 ng of DNA in a 10 µLtotal volume.

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2.2. 1.5 µL of your chosen Rapid Barcode (RB01-24). (1 barcode per sample).

 


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3. Mix by gently by stirring or pipetting until thoroughly mixed. If there are any bubbles present, spin down briefly (2 s-3 s) to ensure all liquid is at the bottom. 

 


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4. Incubate the tubes in the thermal cycler (PCR machine) at 30 °C for 2 min then 80 °C for another 2 min.

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5. Briefly place the tubes on ice to cool.

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6. Pool all your samples into a 1.5ml clean eppendorf DNA LoBind tube. Note down the total volume after pooling. It should be 11.5 µLmultiplied by the number of samples.

 



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7. Only 1 person is required to carry out the following steps.

Resuspend the Pronex beads by vortexing for 10 s or longer.

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8. Use a 1:1 ratio of sample to Pronex beads and mix into the sample by slowly pipetting 10 times. 

 


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9. Incubate the sample at Room temperature for 10 min.

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10. To do while waiting
Take out Elution Buffer (EB) to thawon ice.

Place the sample on a magnetic stand for 2 minuntil the solution becomes clear and the beads form a pellet on one side of the tube.

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11. While leaving the tube on the magnet, carefully remove and discardsupernatant without disturbing the beads.
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12. Wash 1: While still on the magnetic stand, carefully add 200 µL of Pronex wash buffer without flushing directly onto the pellet. If 200 µL is not enough to submerge the pellet, use more wash buffer.

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13. Allow to incubate for 30 s-1 min .

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14. While leaving the tube on the magnet, carefully remove and discard supernatant without disturbing the beads.
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15. Wash 2: While still on the magnetic stand, carefully add 200 µLof Pronex wash buffer without flushing directly onto the pellet. If 200 µL is not enough to submerge the pellet, use more wash buffer.

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16. Allow to incubate for 30 s-1 min .

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17. While leaving the tube on the magnet, carefully remove and discard supernatant without disturbing the beads.
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18. Allow the sample to air dry with lids open for 2 min-5 min watching it until the pellet is no longer shiny.


 


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19. Remove the tube from the magnetic rack and add 15 µL of elution buffer (included in the Nanopore Rapid Barcoding kit). Resuspend the beads by slowly pipetting or stirring with the pipette tip. 

 


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20. Incubate the samples at Room temperature for 10 min to elute the DNA.

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21. Return the tube to the magnetic stand for 1 minuntil the solution becomes clear and the beads form a pellet. 

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22. Store the Pronex beads in the fridge.
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23. Transfer 11 µL of the eluate into a clean 1.5ml Eppendorf DNA LoBind tube.


 



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24. Take another 1 µLof the elute from the tube on the magnetic stand for quantification on a fluorometer (Qubit or Quantus). The remaining beads can be kept in a closed tube on ice, for re-elution, if necessary.

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25. In a new 1.5 mL Eppendorf DNA LoBind tube, mix the following:

1.5 µLRapid Adapter (RA)
3.5 µL Adapter Buffer (ADB)

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26. Add 1 µL of this RA + ADB mixture to the DNA.

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27. Mix gently by flicking and spin down briefly (2 s-3 s).

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28. Incubate this for30 min atRoom temperature.

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29. Preparing the flowcell

Remove the following Nanopore Rapid Kit (RBK-114.24) items from the -20 °Cfreezer, spin down and store on ice.

SB (Sequencing Buffer)
LIB (Library Beads)
FCT (Flow Cell Tether)
FCF (Flow Cell Flush)
Bovine Serum Albumin (BSA) at 50mg/ml

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30. Prepare the flow cell Priming Mix in a fresh 1.5 mL Eppendorf DNA LoBind. Mix by inverting the tube.

1170 µL FCF
5 µL BSA (Bovine Serum Albumin)
30 µL FCT


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31. Remove the flow cell you want to use and slide it under the metal clip in the Mk1B or Mk1C MinION. Press down firmly to ensure correct contact with the thermal and electrical connections.

Mk1B: Plug in the MinION Mk1B to a laptop with Minknow software
Mk1C: Turn on the MinION Mk1C.
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32. Complete a flow cell check to assess the number of pores available on the flow cell.
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33. Rotate the flow cell priming port cover clockwise to open the priming port.
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34. After opening the priming port there will be a small air bubble under the cover that needs to be removed.

Set a P1000 pipette to 200 µL
Insert the tip into the priming port
Turn the adjustment wheel slowly, pausing every few μls,until the pipette shows 220 µL - 230 µLto draw a total of 20 µL-30 µL out of the priming port, or until you can see a small volume of liquid entering the pipette tip.

 




 


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35. Load slowly 800 µLof the Priming Mix from Step 30 into the priming port, without introducing any bubbles. Leave for 5 min and while waiting, proceed to the next step.

 



 


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36. Mix the LIB (Library Beads) thoroughly by pipetting. 
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37. In a new 1.5 ml LoBind tube, mix the following to prepare your library:

37.5 µL SB (Sequencing Buffer)
25.5 µL LIB (Library Beads), mixed immediately before use
12 µL of DNA library (your sample)


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38. Gently open the SpotON sample port cover
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39. Load slowly 200 µL of the Priming Mix into the priming port (not the SpotON sample port). Again, avoid introducing any air bubbles.


 



 

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40. Mix the prepared library by gently pipetting just prior to loading drop by drop 75 µLof the prepared library from Step 37 onto the SpotON sample port. Let each drop flow into the port before adding the next drop.

 


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41. Replace the SpotON sample port cover and gently press down to ensure the bung is in the port.
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42. Rotate the Priming port cover back to close the Priming port.
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43. Quickly cover the lid of the MinION to protect from light.
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44. Start the run on the Minknow software interface.