One Step SYBR Green RT-qPCR Premix HYA416

Components

*The components already include dNTP Mixture, Mg²⁺, ROX Passive Reference Dye, etc.

**One Step SYBR Green RT-qPCR Premix contains RTase, RNase inhibitor and Taq DNA Polymerase in optimized ratios.

Storage

This product should be stored at -20℃ for long-term storage and should be protected from light.

Instructions

1. qRT-PCR ReactionSystem

*Generally speaking, a final primer concentration of 0.2 μM in the reaction system can achieve a good enhancement effect. When the reaction performance is relatively poor, the primer concentration can be adjusted within the final concentration range of 0.1~1.0 μM.

**The accuracy of the final amount of template added when establishing the reaction system will have a great impact on the quantitative results. Therefore, it is recommended to add the template to the reaction system later (such as leading to 2 - 5 μL/sample), which can effectively improve the repeatability of the experiment. It is recommended to use a total RNA template of 1 pg - 1 μg.

2. qRT-PCR Reaction Program

*For templates with complex secondary structures or high GC regions, the reverse transcription temperature can be increased to 55°C or 60°C, which helps to improve subsequent efficiency and; the reverse transcription time can be extended to 15 minutes, which helps to increase cDNA yield.

*This pre-denaturation condition is suitable for existing reactions. If the template structure is complex, the pre-denaturation time can be extended to 3 minutes to improve the pre-denaturation effect.

Technical Information

1. Template diluent selection: You can use purchased Nuclease-free water, DEPC water, or laboratory-prepared nuclease-free water to dilute the template. TE solution contains EDTA, which will inhibit the activity of the enzyme and cannot be used as a diluent.

2. SYBR Enzyme mix contains a high concentration of glycerol. Please centrifuge briefly before use, collect it to the bottom of the reaction tube, and gently aspirate it with a pipette. Mix it thoroughly and then accurately aspirate it.

3. When designing primers, the recommended target PCR product length is 80~150 bp. Primers that have not been used for a long time should first be tested for integrity by PAGE electrophoresis to exclude the possibility of degradation.

4. When the template-free negative control shows obvious amplification, the contamination of the reaction system and the aerosol contamination in the preparation environment should be considered. You can replace new Kit, water, and primers to repeat the experiment; the reaction system should be prepared in an ultra-clean workbench to reduce aerosol contamination.

5. When the melting curve has multiple peaks, consider the possibility that the RNA template contains genomic contamination, nonspecific amplification, primer dimers, etc.

6. If you need to improve the specificity of amplification, you can appropriately increase the annealing temperature (increase 3°C each time).

7. If you need to improve the amplification efficiency, you can extend the extension time of the two-step standard program to 60 sec or use the three-step program. The two-step amplification program generally sets the signal acquisition at the 60°C annealing extension stage; thethree-step amplification program should set the signal acquisition at the 72°C extension stage. The program settings are as follows: