This procedure was used for a fungal ITS barcoding project (ITS5-ITS4).
Primers
The forward and reverse primers with sample indexes were mixed to a final concentration of 5 µM (each), diluted with Qiagen elution buffer (10 mM Tris-HCl, pH 8.5).
PCR amplification
Using the GoTaq G2 hot-start green master mix.
| Component | original conc. | target conc. | one reaction | master mix (442 rxn = 4 x 96 + 15%) |
|---|---|---|---|---|
| Master Mix | 2x | 1x | 5 µl | 2210 µl |
| Primer mix | 5 µM (each) | 0.5 µM | 2 µl | |
| DNA | 1 µl | |||
| water | 2 µl | 884 µl | ||
| total | 10 µl | 3094 µl |
First, distribute 7 µl to each well, then add 2 µl forward + reverse primers and 1 µl DNA separately.
The cycling protocol (36 PCR cycles):
| Step | Temperature | Time |
|---|---|---|
| Initial denat. | 95 °C | 2 min |
| Denaturation | 95 °C | 30 s |
| Annealing | 52 °C | 30 s |
| Extension | 72 °C | 75 s |
| Final extension | 72 °C | 5 min |
It is hepful to check the amplification on a gel for at least a few samples before proceeding.
Pooling
- Pooled 1.5 - 6 µl from each amplicon (depending on sample number) into 8-strip PCR tubes, then combined them into a Eppendorf tube.
- Cleanup with AmpureXP beads (0.6:1 ratio works for ITS; 300ul beads + 500ul pool) → eluted in 30-40ul water or elution buffer.
- Dilution to 20 ng/ul = 1µg in 50µl
Library preparation and sequencing
- Following the protocol Ligation sequencing V14 - PCR barcoding guide (or newer), Starting at step 6. End-prep
- Sequencing on a MinION for up to 24h to generate up to 20M reads: this is more than enough for 15 plates, we also obtained good barcode sequences for most samples with only 3M reads.
- Up to 3 runs were done on the same flow cell