ONT Q20+ (V12) Adapter Ligation for Fungal DNA Barcoding

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

AMX H -

Quick T4 Ligase -

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.

LNB -

Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice.

EB - Lig

Thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.

SFB -

In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

Between each addition, pipette mix 10-20 times.

Reagent 9.4.1 Flongle Volume R10.4.1 Volume

DNA sample from the previous step 30 μl 60 μl

Ligation Buffer (LNB) 12.5 μl 25 μl

NEBNext Quick T4 DNA Ligase 5 μl 10 μl

Adapter Mix H (AMX H) 2.5 μl 5 μl

Total 50 μl 100 μ

0h 0m 5s Spin down with a mini centrifuge for 0h 0m 5s.

Incubate the reaction for 0h 10m 0s at room temperature.

Resuspend magnetic bead stock by vortexing.

Add 20µL (Flongle) or 40µL (R10.4) of resuspended beads to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at room temperature.

Spin down the sample for 0h 0m 5s and pellet on a magnet for 0h 2m 0s. Keep the tube on the magnet, and pipette off the supernatant.

Wash the beads by adding 100-250 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down for 0h 0m 5s, then return the tube to the magnetic rack for 0h 2m 0s and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Note: flicking the tube does not seem to fully resuspend the beads. Just flick 10 times or so.

SFB -

Repeat the previous step.

Spin down for 0h 0m 5s 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 15 μl Elution Buffer (EB). Spin down for 0h 0m 5s and incubate for 0h 10m 0s at room temperature.

Pellet the beads on a magnet until the eluate is clear and colorless, for at least 1 minute.

Remove and retain 15 μl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

Store on ice until you are ready to load in your flowcell.

If you have access to a Quantus or Qubit fluorimeter, now is a good time to quantify 1uL of DNA in your sample.

It is recommend loading 5 fmol to 10 fmol of this final prepared library onto your flow cells. Loading more than 20 fmol of DNA can reduce the rate of duplex read capture. Dilute the library in Elution Buffer if required.

https://www.promega.com/resources/tools/biomath/

For 900bp length DNA (what our ITS1F-4 rxns appear to average), we are looking for

Flongle R9.4.1: 5 fmol - 20 fmol = .003ug - .012ug of DNA.

MinION R9.4.1: 5 - 50 fmol = .003ug - .029ug of DNA.

MinION R10.4.1: 25 - 75 fmol = .015ug - .044ug of DNA.

For a 22 ng/uL sample (Quantus quantification):

22ng 1ug

-------- * -------- = 0.022ug/uL * 15uL (elution buffer; or 13 if you quantified using 2uL) = 0.33 ug DNA in sample of 15uL elution buffer

1uL 1000ng

*Note: the 0.33 in the calculations below will change based on your individual DNA amount.

**Also note: The ONT protocol suggests using additional EB in order to make concentration adjustments. As there is not a lot of the reagent in the standard packets, and it is not possible to buy more individually, I have been using molecular water for this step with no ill effect. You could always calculate

Flongle 9.4.1

How much additional molecular water to have 5.5uL needed for the next step give us correct amount of DNA?

0.33ug / xuL = 0.010ug (17 fmol DNA) x = 33uL x 5.5uL = 181.5uL - 15uL = 166.5uL

So at 0.022ug/uL quantification, add an additional 166.5uL of molecular water to have right concentration to use 5.5uL for the next step with Flongle.

11ng/uL sample comes out to adding an additional 78.5 uL of molecular water.

31ng/uL sample comes out to adding an additional 221.65 uL of molecular water.

I would use 150 uL if you are not able to quantify your sample.

MinION R9.4.1

0.33ug / xuL = 0.025ug (42 fmol DNA) x = 13.2uL x 11uL = 145.2uL - 15uL = 130uL elution buffer addition.

MinION R10.4.1

0.33ug / xuL = 0.04ug (67 fmol DNA) x = 8.25uL x 11uL = 90.75uL - 15uL = 75uL elution buffer addition.