2 × Medino® Universal Advanced qPCR SYBR Master Mix (UDG plus)

Product Name2 × Medino® Universal Advanced qPCR SYBR Master Mix (UDG plus)
Cat.No. LM601017
Size5 mL/5×5 mL
Storage and shipping1. The product is shipped with ice pack.

2.  The product can be stored at -15℃ ~ -25℃ for 24 months.

3. The product contains fluorescent dyes, so it is necessary to avoid strong light irradiation when storing or preparing the reaction system.

Description

Product description

This product is a dedicated reagent for qPCR using the SYBR Green Ⅰ chimeric fluorescence method. The core component is a chemically modified hot-start DNA polymerase, combined with a reaction buffer optimized for qPCR, which can effectively inhibit non-specific amplification, significantly improve amplification efficiency, and accurately quantify target genes. At the same time, this product uses UDG enzyme and dUTP to effectively prevent cross-contamination of PCR products and make the data more accurate. This product contains a special ROX Reference Dye, which is suitable for all qPCR instruments. There is no need to adjust the ROX concentration on different instruments. You only need to add primers, template and water when preparing the reaction system to start the reaction.

  1. Use chemically modified hot-start enzymes to improve sensitivity and enhance specificity.
  2. The use of dUTP and UDG enzymes can effectively eliminate cross-contamination of previously amplified products, and the data is more accurate.
  3. The reaction buffer optimized for qPCR enhances specificity, reduces primer-dimer formation, improves amplification efficiency, and has good reproducibility and high reliability.
  4. Contains special ROX Reference Dye, suitable for all qPCR instruments.

 

Components

 

Components

LM601016-01

(500rxn/20μl reaction)

LM601016-02

(2500rxn/20μl reaction)

2 × Medino® Universal Advanced qPCR SYBR Master Mix (UDG plus) a)4×1.25 ml5×LM601017-01
  1. Contains hot start enzyme, UDG enzyme, dNTPs, dUTP, Mg2+, SYBR Green I, Specific ROX  Reference  Dye etc.

 

Operate

 

qPCR reaction System

ComponentsVolume μLFinal Conc.
2 × Medino® Universal Advanced qPCR SYBR Master Mix (UDG plus)101 ×
Forward Primer (10 μmol/L)0.40.2 μmol/L
Reverse Primer (10 μmol/L)0.40.2 μmol/L
DNA/cDNA templateVariableAs Required
ddH20Up to 20


[Note]:  The amount of each component in the reaction system can be adjusted according to the following principles:

  1. Primer concentration: The final primer concentration is 0.2 μmol/L, and can also be adjusted between 0.1 and 0.5 μmol/L as appropriate.
  2. qPCR is extremely sensitive. The accuracy of the amount of template added when establishing the reaction system will have a great impact on the final result. It is recommended to dilute the template before adding it to the reaction system, which can effectively improve the repeatability of the experiment.
  3. If the template is undiluted cDNA stock solution, the volume used should not exceed 1/10 of the total qPCR reaction volume.

Reaction program

Cycle stepTemp.TimeCycles
Enzyme activation95℃3 min1
Denaturation95 °C5 sec40
Annealing/Extensiona)60 °C30 sec
Melting curve stageb)As Required1

[Note]:

  1. Please adjust the annealing/extension time according to the minimum data collection time limit required by the real-time PCR instrument you are using: at least 30 seconds when using ABI 7700 and 7900; at least 31 seconds when using ABI7000 and 7300; at least 34 seconds when using ABI7500.
  2. The melting curve collection program varies depending on the instrument type. Use the default melting curve collection program of the instrument.

Notes

  1. Avoid repeated freezing and thawing of this product to prevent enzyme activity from decreasing. If the amount used each time is small, it is recommended to store it in aliquots.
  2. Before use, mix the premix by turning it upside down. The premix can be used after instant centrifugation.
  3. When 2× Medino® Universal Advanced qPCR SYBR Master Mix (UDG plus) is stored at -20℃, precipitation may sometimes form. After a short period of storage at room temperature, vortex mixing can completely dissolve. Ensure that the reagents are mixed evenly before use.
  4. Since this product contains the fluorescent dye SYBR Green I, strong light exposure should be avoided as much as possible when storing the premix and preparing the reaction system.
  5. Since this product has extremely high detection sensitivity, please use clean sterilized gun tips and reaction tubes during the preparation process. Laboratories with conditions permitting recommend the use of dedicated pipettes to avoid contamination.
  6. This product is for research use only.

 

Questions and Solutions

 

  1. Signals are generated in the negative control
  2. Template or reagents are contaminated with nucleic acids: Take standard precautions before performing PCR reactions to reduce the risk of contamination.
  3. Primer dimers are generated: Analyze with melting curves.
  1. Multiple peaks appear in the melting curve
  2. Primer dimers or other special structures exist: Design and synthesize new primers according to the design principles
  3. Primer concentration is too high: Reduce primer concentration appropriately
  4. cDNA template contains genomic contamination: Re-prepare cDNA template
  1. No amplification curve
  2. Insufficient reaction cycles: Generally, the number of cycles is set to 40.
  3. Confirm whether the signal acquisition step is set in the program: The two-step amplification program generally sets the signal acquisition in the annealing extension stage.
  4. Confirm whether the primer is degraded: Primers that have not been used for a long time may degrade. Synthesize new primers and repeat the experiment.
  5. The template concentration is too low: Reduce the dilution and repeat the experiment. Generally, samples with unknown concentrations are tested at the highest concentration.
  6. Template degradation: Re-prepare the template and repeat the experiment.
  1. The quantitative PCR amplification curve is not smooth
  2. The signal is too weak: increase the template concentration and repeat the experiment
  3. The wrong ROX type is used: confirm whether the ROX used matches the machine model
  4. Volume changes during the quantitative PCR reaction: the PCR tube is not tightly covered, causing the reaction system to evaporate
  1. Ct value appears too late
  2. Low amplification efficiency: optimize reaction conditions, or redesign synthetic primers
  3. Template concentration is too low: reduce dilution and repeat the experiment. Generally, samples of unknown concentration are tested at the highest concentration
  4. Template degradation: re-prepare template and repeat the experiment
  5. PCR product is too long: the recommended PCR product length is 80-150 bp
  6. PCR inhibitors exist in the reaction system: generally introduced by template, increase template dilution multiple or re-prepare template, repeat the experiment
  1. Poor experimental repeatability
  2. Inaccurate sample volume: use an accurate pipette; increase the template dilution and add a large volume to the reaction system
  3. Template concentration is too low: the lower the template concentration, the worse the repeatability. Reduce the template dilution or increase the sample volume
  4. Volume changes during quantitative PCR reaction: PCR tubes are not tightly covered, resulting in evaporation of the reaction system
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