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 |
AppliedBiosystems: 7500,7500 Fast,ViiA7,QuantStudio Dx, QuantStudio3 and 5, QuantStudio6,7,12k Flex;
Stratagene: MX3000P, MX3005P, MX4000.
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.
Table 1. Reaction Setup
Component | Volume (μl) | Volume (μl) | Final Concentration |
---|---|---|---|
qPCR SYBR Green Master Mix (low ROX) | 25 | 10 | 1× |
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 |
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.
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.
Please follow the instructions