Metabarcoding PCR Protocol

Label PCR tubes and place them under a UV light for 0h 10m 0s.

Add 10µL of Invitrogen Platinum Hot Start PCR 2X Master Mix (REF 13000014) buffer and 8µLof molecular water to a 1.5 mL tube PER NUMBER OF SAMPLES i.e., for 4 samples, add 40 uL of buffer and 32 uL of molecular water to a 1.5 mL tube.

Mix well using a vortex, then centrifuge.

Transfer 18µL of the master mix to each PCR tube.

Transfer 5µL of the DNA template to each PCR tube.

Transfer 2µL of the Metabarcoding Primer to the assigned PCR tube. Immediately place primers on a PCR Cooler Rack after use.

Mix the samples well using a vortex, then briefly centrifuge them using a microcentrifuge or platefuge to ensure all contents are at the bottom.

Place samples in a thermo cycler and set to a PCR protocol. Set the thermo cycler to use a heated lid and wait until the lid and block are close to the temperature. See 8.1 for thermo cycler specifications.

Tip: In the Bik Lab, select protocol “METAB-HS” on thermo cycler.

METAB-HS Protocol:

Lid: 105°C

Volume: 25 μL

1. 94°C, 3:00

2. 94°C, 0:45

3. 50°C, 1:00

4. 72°C, 1:30

5. GOTO step 2, 34X

6. 72°C, 10:00

7. 4°C, ∞

Close the thermo cycler lid well.