Two-Step qRT-PCR Kit
This kit is a qRT PCR kit that integrates cDNA first strand synthesis reagent (RT kit) and ready-to-use qPCR reagent. The first strand synthesis reaction and qPCR reaction of cDNA were carried out in two centrifuge tubes, respectively.
Kit features:
1. Ready to use, sufficient for 50 RT reactions (10 μ L system) and 50 qPCR reactions (20 μ L system).
2. RT and PCR are conducted separately, facilitating the optimization of RT and qPCR conditions, and also facilitating the use of one RT reaction for qPCR amplification with multiple different primers.
3. RT mix contains RT components other than enzymes, templates, and primers, simplifying the reaction setup steps.
4. qPCR mix is 2 × The pre prepared solution also simplifies the reaction setup steps.
5. qPCR mix contains qPCR dyes and does not require the addition of relevant fluorescent PCR dyes.
6. High amplification efficiency, capable of amplifying up to 5 kbp of RNA.
7. Provide a dedicated template diluent for quantitative PCR to ensure the accuracy of low concentration samples during the production of standard curves.
Kit composition:
Component specifications
two × RT Mix 40 μ L (yellow cover)
MMLV reverse transcriptase (including RI) 10 μ L (red cap)
two × QPCR MagicMix 1mL (brown tube)
Quantitative PCR specific template diluent 1mL (green cap)
RNase free water 1mL (blue cap)
Storage condition: -20 ℃, valid for one year.
Self prepared reagents: sample RNA, PCR primers, ROX dyes
Usage:
1、 Preparation of sample RNA (relevant reagents are not provided in this kit)
1. RNA can be extracted from samples using various RNA extraction methods from our company, or products from other suppliers can be selected, but the obtained RNA must be dissolved in RNase free ultrapure water.
If it is necessary to prepare standard curves using RNA, the RNA samples need to be diluted with a quantitative PCR specific template diluent at different gradients in this step, and used as templates for the next RT reaction.
3. Due to the serious impact of contaminated gDNA on RNA quantification, it is necessary to completely remove gDNA contamination from RNA samples. Users can purchase DNase (RNase free) to digest RNA samples or synthesize primers that only recognize exons.
2、 RT (reverse transcription) reaction to synthesize cDNA
4. Prepare the RT reaction system according to the following table (10 μ L system), adding the following components to each tube:
Amount of added substance
Total RNA 100-500ng
two × RT mix 4 μ L
MMLV reverse transcriptase 1 μ L
RNase free water replenishment to 10 μ L
Heat preservation at 5.42 ℃ for 60 minutes. This step is an RT reaction.
Hold at 6.85 ℃ for 5 seconds to terminate the reaction, and then place it on ice for further use.
The synthesized cDNA can be directly used as the next qPCR template without the need for purification. The remaining samples can be stored at -20 ℃ for a long time. if necessary