Protocol Citation: Teddy Urvois, Anne-Laure Besnard, Erwan Quéméré 2024. TaqMan qPCR INRAE eWHALE . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3xj87g25/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: February 15, 2024
Last Modified: May 28, 2024
Protocol Integer ID: 95292
Abstract
Protocol for the TaqMan assay used by INRAE team in eWHALE ring test study
Materials
- Pipettes : monochannel p20, p100, p200, p1000, multichannel p20 and corresponding filter tips
- 1.5 mL microcentrifuge tubes
Before start
Lab work:
Put all equipment (micropipettes, disposable tips, microtubes, molecular grade water, qPCR plate) in UV cabinet and
light UV for 20 mins.
Thaw, vortex and spin samples.
Thaw and vortex Master Mix environnemental TaqMan‱ 2.0.
Master mix preparation
Master mix preparation
Determine the number of reactions, including negative controls and NTCs. Add 2-3 reactions to have margin for pipetting error.
Prepare the Master mix according following this volume for 1 reaction :
4 µL of Molecular Grade Water
10 µL Master Mix environnemental TaqMan‱ 2.0
1 µL Probe at 0.13 micromolar (µM)
1 µL Forward primer at 0.5 micromolar (µM)
1 µL Reverse primer at 0.5 micromolar (µM)
Vortex and spin.
Plate preparation
Plate preparation
2m
Add 17 µL of Master mix.
Add 3 µL of Sample .
Seal the plate with qPCR plate seal and centrifuge briefly (00:02:00 at 2000g).
2m
qPCR
qPCR
11m 20s
Put the plate in the thermal cycler. qPCR is performed with an initial denaturation at 95 °C for 00:10:00 followed by 49 cycles of 00:00:30 at 95 °C and 00:00:50 at 60 °C .
11m 20s