5 × Hifair™ RT-qPCR Buffer is the buffer component of the Hifair™ Multiplex One-Step RT-qPCR Probe Kit (UDG Plus) (Cat# 16630). When combined with the corresponding enzyme mix, it enables multiplex quantitative PCR directly from RNA templates.

In this workflow, reverse transcription and qPCR are performed sequentially in a single tube, streamlining the procedure and minimizing the risk of contamination.

Components

Notes

1. This product is for research use only.

2. Please operate with lab coats and disposable gloves, for your safety.

Instructions

1. Preparation of reaction system((25 µL Example):

[Note]: Thoroughly mix all reagents before use. Avoid vigorous pipetting or shaking to prevent excessive bubble formation.

1. Primer Concentration:

The Primer Mix contains multiple primer pairs. The final concentration of each primer can typically be adjusted between 0.1–1.0 μM, depending on assay requirements.

2. Probe Concentration:

The Probe Mix includes multiple probes labeled with distinct fluorophores. The final concentration of each probe can be optimized within the range of 50–300 nM.

3. Template Dilution:

Due to the high sensitivity of qPCR, template dilution is strongly recommended to achieve optimal Ct values in the range of 20–35.

4. Reaction Volume:

A reaction volume of 25 μL or 50 μL is recommended to ensure robust amplification and reproducibility of target genes.

5. Preparation of reaction system:

1) Prepare the master mix in a biosafety cabinet or clean workspace.

2) Use nuclease-free pipette tips and PCR tubes.

3) Filtered (aerosol-resistant) tips are highly recommended.

4) Take precautions to avoid cross-contamination and aerosol carryove