HotScript One-Step RT-PCR Kit
This kit is specially prepared for one-step RT-PCR experiments, and can be used to conveniently and quickly complete reverse transcription and PCR amplification reactions in the same reaction tube. During the reaction process, there is no need to open the tube cap to add reagents, which avoids contamination and improves detection sensitivity and experimental efficiency.
This kit includes multiple point mutation modified heat-resistant HotScript M-MuLV reverse transcriptase, hot start Taq DNA polymerase, and RNasin, as well as an optimized unique reaction system suitable for reverse transcription and PCR amplification.
The RNase H activity of HotScript M-MuLV reverse transcriptase is missing, which has stronger elongation and stability compared to M-MuLV. It can be used for longer cDNA synthesis and the construction of a high proportion of full-length cDNA libraries. At the same time, this enzyme can be reverse transcribed at temperatures as high as 50-60 ℃, greatly improving the complex secondary structure. The GC content is rich, and the template reverse transcription efficiency is significantly higher. The yield sensitivity and specificity are significantly higher than those of general kits.
Applicable scope:
Suitable for high copy and low copy gene detection;
Suitable for RNA templates with high GC content or complex secondary structures.
Kit composition:
Component 50 μ L × 50 times 50 μ L × 100 times
two × RT-PCR Buffer 625 μ L 1250 μ L
Enzyme Mix 25 μ L 50 μ L
HotScript M-MuLV RTase 25 μ L 50 μ L
RNase free H2O 1.5mL 1.5mL
Operating steps:
two × RT-PCR Buffer may have crystallization precipitation at low temperatures, and should be used after heating the palm or vortex oscillation to restore melting. Short term use can be stored in a 4 ℃ refrigerator.
Configure reaction system:
Component dosage and final concentration
two × RT-PCR Buffer 25 μ L 1 ×
Forward Primer (10 μ m) 1 μ L 0.2 μ M
Reverse Primer (10 μ m) 1 μ L 0.2 μ M
Enzyme Mix 1 μ L-
HotScript M-MuLV RTase 1 μ L-
RNA template X μ 1pg-2 μ G
RNase free H2O to 50 μ L-
Attention: The primer concentration should be between 0.2 and 0.6 at the final concentration μ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the concentration of primers can be reduced to optimize the reaction system. If multiple reactions are carried out at the same time, first prepare a mixture in proportion, shake and mix well, and then press 50-X per tube μ L (X is the template quantity) for packaging.
Mix and centrifuge the prepared reaction system.
Preheat the thermal cycler to 50-55 ℃, place the PCR tube in the thermal cycler, and perform the reaction according to the following reaction conditions.
Step Temperature Time Cycles
1 50~55 ℃ 30 minutes
