TaqMan qPCR INRAE eWHALE
Anne-Laure Besnard, Teddy Urvois, Erwan Quéméré
Abstract
Protocol for the TaqMan assay used by INRAE team in eWHALE ring test study
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 TaqManTM 2.0.
Steps
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 :
4µL of Molecular Grade Water
10µL Master Mix environnemental TaqManTM 2.0
1µL Probe at 0.13micromolar (µM)
1µL Forward primer at 0.5micromolar (µM)
1µL Reverse primer at 0.5micromolar (µM)
Vortex and spin.
Plate preparation
Add 17µL of Master mix.
Add 3µL of Sample.
Seal the plate with qPCR plate seal and centrifuge briefly (0h 2m 0s at 2000g).
qPCR
Put the plate in the thermal cycler. qPCR is performed with an initial denaturation at 95°C for 0h 10m 0s followed by 49 cycles of 0h 0m 30s at 95°C and 0h 0m 50s at 60°C.
