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Kits SYBR Green qPCR Master Mix (Low ROX)

SYBR Green qPCR Master Mix (Low ROX)

Catalog No. C0008
Excel PDF

Description

SYBR Green qPCR Master Mix (Low ROX) is a 2× concentrated, ready-to-use supermix optimized for dye-based quantitative PCR. It contains aptamer-modified hot-start Taq DNA polymerase, dNTPs, Mg2+, SYBR Green I dye, Low ROX, stabilizers. When used, only templates, primers and sterile ultra-pure water are needed to enter the Real Time PCR reaction, which is easy to operate and reduce the probability of contamination.

Pack Size Availability Price/USD Quantity
1 ml (100 rxns) In stock 30.00
1 ml * 5 (500 rxns) In stock 117.00
1 ml * 20 (2000 rxns) In stock 379.00
Bulk Size: Please Quote for Discount

Applications

  • Dye-based quantitative PCR detection
  • Nucleic acid amplification and expression profiling
  • Quantitative genotyping studies
  • Genomics-related applications

Instrument Compatibility

AppliedBiosystems: 7500,7500 Fast,ViiA7,QuantStudio Dx, QuantStudio3 and 5, QuantStudio6,7,12k Flex;
Stratagene: MX3000P, MX3005P, MX4000.

Storage and Stability

  • Stored at -20℃ avoiding the light, valid for 20 months.
  • Please try to avoid repeated freezing and thawing. If frequent use is required, please aliquot in advance.
  • SYBR Green I, a fluorescent dye, is contained in the product. Strong light exposure should be avoided when preserving or preparing the reaction system.

Protocol

Note: White precipitation may appear during the thawing of the qPCR SYBR Green Master Mix. Before use, dissolve the precipitation by incubating at 4℃ and gently flipping the tube. Mix the solution thoroughly every time before pipetting.

  1. Prepare enough assay master mix for all reactions on ice by adding all required components, except the DNA template, according to Table 1.
  2. Mix and dispense equal aliquots into the wells of qPCR plate.
  3. Add DNA samples to each well of qPCR plate. Seal the reaction plate and centrifuge briefly.
  4. Program the thermal cycling protocol on a real-time PCR instrument according to Table 2.
  5. Data analysis.

Table 1. Reaction Setup

Component Volume (μl) Volume (μl) Final Concentration
qPCR SYBR Green Master Mix (low ROX) 25 10
Forward Primer (10μM)1 Variable Variable 0.1–1 μM
Reverse Primer (10μM)1 Variable Variable 0.1–1 μM
DNA template2 As required As required
ddH2O to 50 to 20
  • The final concentration of each primer is usually 0.2 μM.
  • The volume of template (i.e. undiluted cDNA generated from total RNA) should be ≤ 1/10 of total volume. To improve experimental repeatability, it is recommended to dilute the template and pipet 2 μl-5 μl to the reaction system.

Table 2. Thermal cycling protocol (2-Step cycling protocol)

Stage Temperature Time Cycles
Pre-denaturation 95℃ 5 min 1
Denaturation 95℃ 10 sec 40
Annealing/Extension and Plate Read 60℃ 30 sec★
Melting Curve According to instrument guidelines


Table 3. Thermal cycling protocol (3-Step cycling protocol)

Stage Temperature Time Cycles
Pre-denaturation 95℃ 5 min 1
Denaturation 95℃ 10 sec 40
Annealing 55~60℃ 20 sec
Extension and Plate Read 72℃ 20 sec★
Melting Curve According to instrument guidelines


Note: Two-step method can be selected for high specificity, and three-step method can be selected for high-efficiency amplification. A) Pre-denaturation time: it can be appropriately reduced to 2 min according to the specific conditions of different templates and primers. B) Annealing temperature and time: please adjust according to the length of primer and target gene. C) Fluorescence signal acquisition (★): Please set the experimental procedure according to the instructions of the instrument. The time setting of several common instruments is as follows: 30 sec above: Applied Biosystems: StepOne, StepOne Plus,7500 Fast; LightCycler 480; Roche Applied Science: LightCycler 480; Bio-Rad: CFX96 31 sec or above: Applied Biosystems: 7300 34 sec above: Applied Biosystems: 7500 D) Melting curve: The default procedure of the instrument can be used under normal circumstances.

Attention Points in Operation (Please Read Carefully)

  • Avoid repetitive freeze-thaw cycles to prevent polymerase activity from decreasing. Aliquot the mix into small batches for frequent usage.
  • Gently invert the tube upside down several times before use. DO NOT vortex. Brief centrifugation prior to use is recommended.
  • Keep the mix from bright light during storage and usage due to the fact that the fluorescent SYBR Green I dye may fade under light over time, resulting in a decrease in performance sensitivity.
  • Due to the high sensitivity nature of the qPCR reaction, contamination of air or aerosols may lead to reaction failure or result inaccuracy. Please set up the qPCR reaction in a clean environment using filtered tips, and sterilized tubes and pipette sets.

Data analysis

Amplification curve: Generally, the ranges of CT values are 15 to 35, while 20-28 are the best. If the CT value is too low, increase the dilution ratio of template. If the CT value is too high, raise the concentration of templates or primers, even adjust the qPCR program. Melting curve: Usually only when the melting curve is single peak, the quantitative result can be qualified. If there are multiple peaks in the dissolution curve, it is necessary to optimize the conditions, such as redesign primers.

Attention

  • SYBR Green Master Mix (Low ROX) stored at -80℃ may produce white or yellowish precipitation, which can be dissolved slowly by hand, placed at room temperature for a short time away from light, and mixed upside down gently until the precipitation disappears completely, without affecting the reagent performance.
  • Before use, please mix it upside down and upside down gently to avoid bubbles and poor reaction effect due to uneven mixing.
  • Before use, please mix it upside down and upside down gently to avoid bubbles and poor reaction effect due to uneven mixing.
  • This product is only used for scientific research by professionals and shall not be used for clinical diagnosis or treatment. It is strictly prohibited to use in food or medicine.

Please follow the instructions