Jun 20, 2024

Public workspaceMetabarcoding PCR Protocol

DOI
dx.doi.org/10.17504/protocols.io.6qpvr8842lmk/v1
  • 1University of Georgia;
  • 2Department of Marine Sciences, University of Georgia;
  • 3UGA;
  • 4University of Georgia- Holly Bik
Jenna Brown
Open access
DOI: dx.doi.org/10.17504/protocols.io.6qpvr8842lmk/v1
Protocol Citation: Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell, Jenna Brown 2024. Metabarcoding PCR Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8842lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 30, 2024
Last Modified: June 20, 2024
Protocol Integer ID: 100934
Funders Acknowledgement:
Holly Bik
Abstract
18S Metabarcoding PCR Protocol using Invitrogen Platinum Hot Start PCR 2X Master Mix (REF 13000014) buffer.
Preparing the PCR tubes
Preparing the PCR tubes
Label PCR tubes and place them under a UV light for Duration00:10:00 .

10m
Preparing the master mix
Preparing the master mix
Add Amount10 µL of Invitrogen Platinum Hot Start PCR 2X Master Mix (REF 13000014) buffer and Amount8 µL of 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.
Metabarcoding PCR
Metabarcoding PCR
Transfer Amount18 µL of the master mix to each PCR tube.

Transfer Amount5 µL of the DNA template to each PCR tube.

Transfer Amount2 µL 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.