Medino® qPCR SYBR Green Master Mix(High Rox Plus)

Cat.No. LM601005
Size1mL/5×1mL/50×1mL/100×1mL
Storage and shippingStore at -25 to -15℃ with a shelf life of 18 months. Avoid repeated freeze-thaw cycles. This product contains the fluorescent dye SYBR Green I, so it should be protected from exposure to strong light during storage or preparation of reaction systems.
Compatible Models

 

Applied Biosystems: 5700, 7000, 7300, 7700, 7900HT FAST, StepOne, StepOne Plus.
Category:

PDF Download: LM601005

 

Product description

Medino® qPCR SYBR Green Master Mix (High Rox Plus) is a 2× pre-mixed solution for real-time quantitative PCR amplification. The mix contains thermostable HieffoDNA Polymerase, SYBR Green I, dNTPs, Mg2+, and HighRox. When used, simply add template and primers to the amplification system to perform real-time fluorescence quantitative PCR, greatly simplifying the operation process and reducing the risk of contamination.

The DNA polymerase ligand used in this product can dynamically adjust the activity of DNA polymerase with temperature changes. The formula is supplemented with factors that effectively inhibit non-specific PCR amplification and enhance PCR reaction efficiency, enabling quantitative PCR to achieve good linear relationships across a wide range of quantitation.

 

Instructions for Use

 

  1. qPCR reaction System

 

Components Volume μL****Volume μLFinal Conc.
Medino® qPCR SYBR Green Master Mix (High Rox Plus)*25101 ×
Forward Primer (10μM)**10.40.2 μM
Reverse Primer (10 μM)**10.40.2 μM
DNA/cDNA template***xx
ddH2Oto 50to 20

[Note]:

* Before use, thoroughly mix to avoid excessive bubble formation caused by vigorous shaking.

** The typical final concentration of primers is 0.2 μM, but it can be adjusted between 0.1-1.0 μM according to the situation.

*** If the template is undiluted cDNA stock, the volume used should not exceed 1/10 of the total qPCR reaction volume. It is recommended to dilute the cDNA stock 5-10 times, and the optimal template input should yield a CT value between 20-30 cycles.

**** It is recommended to use 20 μL or 50 μL to ensure the effectiveness and reproducibility of gene amplification.

***** Please prepare in a laminar flow hood and use pipette tips and reaction tubes free of nucleic acid residues; it is recommended to use filter-tipped pipette tips to avoid cross-contamination and aerosol contamination.

 

 

  1. Reaction program
  • Two-step amplification program*
Cycle stepTemp.TimeCycles
Denaturation**95 °C5 min1
Amplification95 °C10 sec40
60 °C***30 sec****
Melting curveDefault setting on instrument1

 

  • Three-step amplification program
Cycle stepTemp.TimeCycles
Denaturation**95 °C5 min1
Amplification95 °C10 sec40
55 – 60 °C***20 sec****
72 °C20 sec****
Melting curveDefault setting on instrument1

[Note]:

* For high specificity, you can choose the two-step method; for high-efficiency amplification, you can choose the three-step method.

** The denaturation time can be shortened to 2 minutes depending on the specific template and primers.

*** Adjust annealing temperature and time according to the length of primers and target genes.

**** For fluorescence signal collection, please follow the experimental program setting requirements in the instrument manual. The time settings for several common instruments are as follows:

30 seconds or more: Applied Biosystems: StepOne, StepOne Plus, 7500 Fast; Roche Applied Science: LightCycler 480; Bio-Rad: CFX96

31 seconds or more: Applied Biosystems: 7300

34 seconds or more: Applied Biosystems: 7500

 

  1. Result Analysis

Quantitative experiments require at least three biological replicates. After the reaction, it is necessary to confirm the amplification and melting curves.

  • Amplification Curve: The standard amplification curve should exhibit an S-shape.
  • Ct values falling between 20-30 are most accurate for quantitative analysis.
  • Ct values less than 10 indicate the need for template dilution and repeating the experiment.
  • Ct values between 30-35 suggest the need to increase template concentration or enlarge reaction volume to improve amplification efficiency and ensure accurate analysis.
  • Ct values above 35 render the results unsuitable for quantitative analysis but may be used for qualitative assessment.

 

  • Melting Curve:
  • A single peak in the melting curve indicates good reaction specificity suitable for quantitative analysis.
  • Presence of multiple peaks or double peaks in the melting curve indicates unsuitability for quantitative analysis. In the case of double peaks, DNA agarose gel electrophoresis can determine if the non-target peak is due to primer dimers or non-specific amplification. If primer dimers are detected, it is advisable to lower primer concentration or redesign highly specific primers. If non-specific amplification is observed, increase annealing temperature or redesign more specific primers.

 

  1. Primer Design Guidelines
  • Recommended primer length is around 25bp. Optimal amplicon length is 150bp, but lengths between 100bp to 300bp are acceptable.
  • The difference in melting temperature (Tm) between forward and reverse primers should not exceed 2° Primer Tm values between 60°C and 65°C are optimal.
  • The distribution of bases in primers should be uniform, avoiding stretches of four identical bases. The GC content should be around 50%. The last base at the 3′ end is preferably G or C.
  • It is preferable to avoid stretches of three or more complementary bases between internal regions of primers or between forward and reverse primers.
  • Primer specificity should be verified using the NCBI BLAST program. Avoid non-specific complementary sequences of two or more bases at the 3′ end of the primer.
  • Designed primers should undergo efficiency testing, and only primers with similar amplification efficiencies should be used for quantitative comparative analysis.

 

Notes

  1. This product is intended for research use only.
  2. For your safety and health, please wear laboratory attire and disposable gloves when handling.

After thawing, the Master Mix may contain precipitates. Store at 4°C and mix by gently inverting the container until the solution clears. This does not affect the performance of the reagent.

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