One-Step RT-qPCR Probe Kit is a multiplex fluorescence quantitative PCR kit with RNA as a template. During the reaction process, reverse transcription and quantitative PCR are completed in one tube, no additional opening operation is required,
which reduces the risk of contamination. One-Step RT-qPCR Probe Kit selects reverse transcriptase to efficiently synthesize the first-strand cDNA, and selects the optimal ratio of HzymesAccu AM Hot Start Taq DNA polymerase and RNase inhibitor, with additions to inhibit non-specific amplification and improve multiple quantifications.
The amplification efficiency enhancer buffer system, the detection sensitivity can reach 0.1pg, can provide stable and reliable amplification, and can perform up to four-fold reaction. -fold reaction.
a. 2×One Step Buffer contains dNTP Mix、 Mg2＋.
b. Enzyme Mix mainly contains reverse transcriptase, HzymesAccu AM Hot Start Taq DNA polymerase, and RNase inhibitor.
c. ROX: Need to choose calibration according to the type of testing instrument
Transportation and Storage ≤0℃ transportation; -20℃ storage.
Please use an RNase-free pipette tip, EP tube, etc. during the preparation of the experiment.
a. The primer concentration is usually 0.2uM. When the amplification performance is poor, the primer concentration can be adjusted within the range of 0.1-1.0uM according to the situation.
b. Probes containing multiple fluorescent signals can be used, and the final concentration of each probe with different signals can be adjusted between 50nM-300nM.
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 20μL or 50μL for the reaction system.
e. The length of the amplified product is selected in the range of 80-250 bp.
f. Please fully dissolve and absorb before use to avoid excessive bubbles caused by violent shaking.
a. For templates with complex secondary structure or high GC content, it is recommended to increase the reverse transcription temperature to 55°C, which is beneficial to increase the amplification efficiency.
b. The temperature of the amplification reaction can be adjusted according to the Tm value of the primer
c. The time is calculated based on the size of the amplified fragment, generally 1kb/min.