TITLE: FUNDIS Setting up PCR for rDNA ITS Barcoding of Fungi using ONT Nanopore
AUTHORS: Harte Singer

[DESCRIPTION]
This protocol involves using pre-made dual-indexed master mix 96 well plates to run PCR on fungal specimens that have already had their DNA extracted, diluted and in PCR 8 strip tubes. Quality check includes using the BlueGel platform to screen for successful PCR. Adapted from dx.doi.org/10.17504/protocols.io.36wgq7qykvk5/v3

[STEPS]
SECTION: Setting up PCR
2. Remove DNA extracts from the freezer and allow to thaw before beginning. Note the plate number in your notebook (i.e. FDS-CA TB05) 
SECTION: Setting up PCR
3. Once thawed, spin DNA extracts down for 2 s in a benchtop PCR tube centrifuge.

SECTION: Setting up PCR
4. Remove 96 well plate of forward indexed primers (ITS1F) from the freezer and note the index number and primer pair in your notebook (i.e. ONT97 ITS1F ITS4) Remove the foil cover.

SECTION: Setting up PCR
6. Orient yourself so that PCR Strip 1 Tube 1 aligns with well H1 on a standard 96 well plate, and pipette 0.5-1 µL  from each strip into the corresponding row of the 96 well plate using a p10 multichannel pipette.  

Open the PCR strip tubes carefully to avoid splashing the liquid around. Check your fingers to make sure there is no residue from the PCR tubes on them in between opening strips. Wipe any residue off with a paper towel in between opening tube strips or replace gloves as needed. 

Visually inspect the pipette tips for presence of liquid at an approximately equal amount and no large debris before adding to the wells. Gently pipette mix. 

Be extra careful to avoid cross-contamination from drips, splashes, aerosols etc. Gentle, intentional movements and pipetting is necessary.

Choose how much to add based on how much tissue was in the original tube and how dilute it is. In most cases I will use 0.7 µL .

SECTION: Setting up PCR
7. Place a new foil seal on the plate and using a sharpie marker, ensure the seal is tightly attached to the plate. Write the run number using your initials (i.e. HSONT01) The plate number and sampling code ( i.e. FDS-PL01 HS01), the primer pair (i.e. ITS1F ITS4) and the date. Record this information in the notebook and take a photo of the plate for future reference. 

SECTION: PCR Thermal Cycler conditions for ITS Barcoding
10. I have experimented with many different PCR conditions with no noticeable change in amplification efficiency. Touchdown PCR is probably superior. Here are some conditions that should work.  

95 °C 3 min 
Followed by 33 cycles of:
95 °C 30 s 
60 °C 30 s 
72 °C 45 s 
Followed by a final extension
72 °C 90 s 
Hold
12 °C 1 min 

The final hold simply brings the PCR product down in temperature and is not meant to actually hold the PCR product at any particular temperature. It is stable at room temperature for weeks, but should be removed from the machine and placed in the fridge in a plastic ziploc bag within 24 hours. Make sure to turn off the machine. 

SECTION: Setting up PCR
9. Hold the PCR plate in the correct orientation close to your chest and in a single swift but controlled motion, extend your arm away from your chest stopping abruptly when your arm extends. This should dislodge any bubbles from the bottom of the wells of the tube. Do not do this too hard, you definitely do not want to splash the liquid. 
SECTION: Setting up PCR
8. Visually inspect the underside of the plate to see the level of liquid in the tubes. Note any tubes that are obviously low on liquid in the noteboook.
SECTION: Electrophoresis using the blueGel system.
11. Choose 3-5 samples at random from each PCR plate to confirm successful amplification using electrophoresis. Consult the blueGel manual for support. 
SECTION: Setting up PCR
1. Remove pre-mixed green master mix from the freezer, or a tube of indexed primer and a tube of sealed 2x Taq Mean Green Master Mix and allow to thaw for 5 min. If mixing up a new batch, mix a 1:10 ratio of reverse indexed primer to Master Mix (i.e. 140 µLprimer with 1400 µL   Master Mix) note the primer name and index number in your notebook, i.e. ITS4 ONT97

SECTION: Setting up PCR
5. Add 89 µL to each tube in an 8-strip of PCR tubes and then add 6.8 µL of Master Mix to each well of the 96 well plate using a multi-channel pipettor. Use new tips for each addition and do not cross-contaminate.