AffiPCR® All In One 5X RT MasterMix With Genomic DNA Removal
Easy, Efficient, and Reliable cDNA Conversion
Launch your gene expression research studies to new heights with our all-in-one RT-PCR Master Mix. This convenient and pre-optimized solution simplifies the first-strand cDNA synthesis process, saving you time and effort while delivering high-quality results.
Key Features:
- Complete Reaction Mix: Eliminate the need for tedious preparation and component balancing. Our master mix includes all essential reagents for cDNA synthesis:
- Simplified Workflow: Streamline your cDNA synthesis protocol with a one-step setup. Simply add your RNA template to the master mix and run the reaction under optimized thermal cycling conditions.
- High cDNA Yields: Our robust formulation ensures exceptional cDNA yields, ideal for downstream applications like quantitative PCR (qPCR), digital PCR (dPCR), and cDNA library construction.
- Broad Compatibility: The master mix functions seamlessly with a wide range of RNA sources, including total RNA, mRNA, and various cell types.
Benefits:
- Saves Time and Effort: The pre-mixed format eliminates reaction setup time and minimizes errors associated with manual component handling.
- Enhanced Accuracy: Consistent reaction conditions and optimized formulations ensure reliable and reproducible cDNA synthesis.
- Improved Sensitivity: High cDNA yields enable detection of low-abundance transcripts for sensitive downstream applications.
- Cost-Effective: The all-in-one format reduces waste and simplifies purchasing, minimizing research costs.
Applications:
- Gene expression analysis by qPCR and dPCR
- cDNA library construction for next-generation sequencing (NGS)
- In vitro transcription (IVT)
- RNA interference (RNAi) studies
Protocol for cDNA Synthesis using AffiPCR® All in One RT-PCR Master Mix with gDNA Removal
Materials Required:
- AffiPCR® All-in-One RT-PCR Master Mix - Provided
- RNA template (total RNA, mRNA, etc.) - Not Provided
- Nuclease-free water - Provided
- Thermal cycler - Not Provided
- Tubes suitable for PCR - Not Provided
Procedure:
Thaw Master Mix: Remove the All-in-One RT-PCR Master Mix from storage at -20°C or -80°C and allow it to thaw at room temperature. Do not thaw the master mix by heating or microwaving.
Prepare Reaction Mix: In a nuclease-free microcentrifuge tube, prepare the reaction mix by combining the following components:
- AffiPCR® All-in-One RT-PCR Master Mix: 4 μL
- RNA template: 1 ng to 2 μg
- Nuclease-free water: up to 20μL
Mix Thoroughly: Gently vortex or flick the reaction mix tube to ensure thorough mixing of the components. Briefly centrifuge the tube to collect all the liquid at the bottom.
Incubate at 37°C: Place the reaction mixture in a thermal cycler preheated to 37°C and incubate for 15 minutes. This step facilitates the reverse transcription process.
Incubate at 60°C: After the initial incubation, transfer the reaction mixture to the thermal cycler and incubate at 60°C for 10 minutes. This step ensures the completion of the reverse transcription reaction.
Stop the Reaction: If desired, stop the reaction by heating the samples at 95°C for 3 minutes. Chill the reaction mixture on ice to halt any further enzymatic activity.
Storage: Store the synthesized cDNA at -20°C or proceed directly to downstream applications, such as qPCR, dPCR, or cDNA library construction.
Notes:
- Refer to the instructions and guidelines provided with the AffiPCR® All-in-One RT-PCR Master Mix for specific details on reaction setup, cycling conditions, and other recommendations.
- Always use proper laboratory techniques and follow biosafety guidelines while handling RNA and other biological materials.
Recommendations:
- Poly(A) + mRNA and total RNA can be seamlessly utilized for first-strand cDNA synthesis. While total RNA provides a comprehensive snapshot of gene expression, poly(A) + mRNA offers enhanced yields and purity in your final products, ensuring superior results for your downstream applications.
- Elevate the efficiency of genomic DNA (gDNA) removal with a simple adjustment to your protocol. Extend the length of the 37°C incubation from the recommended 15 minutes to 30 minutes for more thorough gDNA elimination. This optimization ensures the purity and accuracy of your cDNA synthesis, paving the way for precise gene expression analysis.