Welcome to the presentation onthe Welcome to presentation

Introduction The q. PCR set enables the simultaneous detection (multiplex) of potato leafroll virus

Suitable tissues for the source of viral RNA are Leaves From tubers: Stolon, peel

The q. PCR set: The q. PCR sets are available for 96 or 192

There are 4 q. PCR kits available. Each kit contains a “rapid extraction set”

Material and Equipment required Rnase-free filter tips and micropipets Optical Rnase-free tubes Disposable latex

Please note! Always wear gloves Use tips with filter …and for your safety

Preparation of the q. PCR samples Gently thaw the kit components on ice or

Preparation of the q. PCR samples II Prepare the master mix reaction by combining

Preparation of the q. PCR samples III Add the reaction mix to each PCR

Thermal cycling Thermal cycle the samples using the following settings:

Monitoring the PCR amplification Monitor the simultaneous PCR amplification with a thermal cycler which

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Welcome to the presentation onthe Welcome to presentation on the q. PCR set/kit for the detection of potato leafroll virus and potato virus Y Dr. Marcel Lüscher Number of Slides: 12 Duration: 4 min by BIOREBA AG 2017

Introduction The q. PCR set enables the simultaneous detection (multiplex) of potato leafroll virus (PLRV) and potato virus Y (PVY) with real time PCR. Viral RNAs extracted from potato samples (with our “potato DNA/RNA rapid extraction set”) are amplified in a one-step RT-PCR reaction. The increase of the amplified c. DNAs can be monitored in real time since the specific probes are labeled with fluorophores.

Suitable tissues for the source of viral RNA are Leaves From tubers: Stolon, peel and sprouts From dormant tubers use: Heel For the procedure of nucleic acid extraction please refer to the Potato DNA/RNA rapid extraction set

The q. PCR set: The q. PCR sets are available for 96 or 192 reactions: Each set contains: Taq Master Mix (2 x) Primers/Probes/IC Mix_PLRV/PVY (10 x) RT Master Mix (50 x) Nuclease free water AND

There are 4 q. PCR kits available. Each kit contains a “rapid extraction set” and a “q. PCR set”: 839610 q. PCR PLRV/PVY kit 96/10: For 96 extractions and a pool size of up to 10 tuber samples 839625 q. PCR PLRV/PVY kit 192/10: For 192 extractions and a pool size of up to 10 tuber samples 839210 q. PCR PLRV/PVY kit 96/25: For 96 extractions and a pool size of up to 25 tuber samples 839225 q. PCR PLRV/PVY kit 192/25: For 192 extractions and a pool size of up to 25 tuber samples

Material and Equipment required Rnase-free filter tips and micropipets Optical Rnase-free tubes Disposable latex or venyl gloves Thermal cycler for real time PCR

Please note! Always wear gloves Use tips with filter …and for your safety

Preparation of the q. PCR samples Gently thaw the kit components on ice or at 4°C. Mix the tubes briefly and spin the liquid down. Determine the number of reactions to prepare, then increase the number by 1 -2 to compensate for pipetting losses.

Preparation of the q. PCR samples II Prepare the master mix reaction by combining the kit components according to the table below:

Preparation of the q. PCR samples III Add the reaction mix to each PCR tube or well of an optical grade plate. Add 2 -5 μl RNA template to the samples reactions. Seal the tubes or plates, centrifuge briefly and protect from light before thermal cycling.

Thermal cycling Thermal cycle the samples using the following settings:

Monitoring the PCR amplification Monitor the simultaneous PCR amplification with a thermal cycler which is able to measure the fluorescence of the following fluorohores: