One-Step RT-qPCR Kit
One Step RT-qPCR Kit is a multiplex fluorescent quantitative PCR kit using RNA as a template. During the reaction, reverse transcription and quantitative PCR are completed in one tube, without additional tube opening operations, reducing the risk of contamination. The One Step RT-qPCR Kit uses reverse transcriptase to efficiently synthesize the first strand cDNA, and selects the optimal ratio of Hot Start Taq DNA polymerase (antibody modification) and RNase inhibitor to add inhibition of non-specific amplification and increase multiplex The enhancer buffer system for quantitative amplification efficiency, the detection sensitivity can reach 0.1 pg, can provide stable and reliable amplification, and can perform up to quadruple reactions.
Note: a. 2×One Step Buffer contains dNTP Mix and Mg2+.
b. Enzyme Mix mainly contains reverse transcriptase, Hot Start Taq DNA polymerase (antibody modification) and RNase inhibitor. ROX: Calibration needs to be selected according to the type of testing instrument.
Transport and storage methods Transport at ≤0°C; store at -20°C. Precautions Please use RNase free pipette tips, EP tubes, etc. during the experimental preparation process. reaction system
a. The primer concentration is usually 0.2 μM. When the amplification performance is poor, the primer concentration can be adjusted within the range of 0.1-1.0 μM according to the situation.
b. Probes with multiple fluorescent signals can be used, and the final concentration of probes for each different signal can be adjusted between 50-300 nM.
c. The sensitivity of qPCR is extremely high. It is recommended to dilute the template and add it to the reaction system. It is appropriate to control the Ct value between 20-35.
d. It is recommended to use 20 μl or 50 μl for the reaction system.
e. Select the length of the amplified product within the range of 80-250 bp.
f. Before use, please fully dissolve and inhale to avoid excessive air bubbles caused by violent shaking.
a. For templates with complex secondary structures or high GC content, it is recommended to increase the reverse transcription temperature to 55°C, which is beneficial to improve the amplification efficiency.
b. The temperature of the amplification reaction can be adjusted according to the average Tm value of the primers and probes being about 5°C lower.
c. If the RT-PCR product is ≥1kb, the cycle step can be increased by one step of 68°C extension, and the time is based on the size of the amplified fragment, generally calculated at 1kb/min