Cat. No.: HY-K0501

SYBR Green qPCR Master Mix

Name: SYBR Green qPCR Master Mix
Brand: MedChemExpress
SKU: HY-K0501
Price: 40.00 USD
Availability: InStock
Rating: 5 (301 reviews)

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MCE qPCR Master Mix is provided as a simple-to-use, stabilized 2× formulation that includes all components for qPCR except sample DNA, primers and water. The 100 rxns is defined as the base specification. All larger sizes correspond to incremental volumes of this base.

Storage :
-20°C, 2 years. Protecting from light, and avoid repetitive freeze-thaw cycles.

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Description & Advantages

The Super Hot-Start SYBR Green qPCR Master Mix is a ready-to-use qPCR cocktail. It contains a novel Taq DNA Polymerase, unique hot start reagents, optimized buffer, SYBR Green I, dNTPs, PCR Enhancer and PCR stabilizer. qPCR Master Mix is provided at 2× concentration and can be used at 1× concentration by adding template, primer, passive reference dye (optional) and ddH₂O.

MCE qPCR Master Mix can be directly used for robust and low-template quantitative PCR with high sensitivity, specificity and reliability. Two separate tubes of ROX Reference Dye are included for use with instruments that require a high or low level of reference dye for rectify the error of fluorescence signals between different wells.

1. Ready to use.

2. Optimized buffer enhances specificity and reduces primer-dimer formation.

3. High sensitivity, specificity and reliability.

4. Passive reference dyes are provided for different qPCR instruments.

Protocol

General Protocol

1. Preparation of the reaction mixture: prepare the reaction mixture on ice. Three technical replicates are recommended for each sample.

Component	50 μL reaction	20 μL reaction	Final concentration
SYBR Green qPCR Master Mix (2×)	25 μL	10 μL	1×
Forward Primer (10 μM)	1 μL	0.4 μL	0.2 μM
Reverse Primer (10 μM)	1 μL	0.4 μL	0.2 μM
DNA/cDNA	4 μL	2 μL	/
ddH₂O	19 μL	7.2 μL	/
Total volume	50 μL	20 μL	/

Note: a. A final primer concentration of 200 nM is commonly used and may be optimized within 0.1-1.0 μM according to amplification efficiency and specificity;
b. Recommended template amount: 10-100 ng genomic DNA or 1-10 ng cDNA. Adjust the template amount according to target abundance to keep Ct values preferably within 15-35;
c. It is recommended to prepare a Master Mix without template first, dispense it, and then add template to reduce pipetting error.

Selection of ROX Reference Dye

Instrument requirement	Use
No ROX normalization required	Do not add ROX Reference Dye
Low ROX required	Use ROX Reference Dye II (50×) at 1× final concentration
High ROX required	Use ROX Reference Dye (50×) at 1× final concentration

Select ROX according to the requirements of the qPCR instrument. When ROX is added, reduce the ddH₂O volume accordingly to keep the total reaction volume unchanged.

2. qPCR cycling program:

Step	Temperature	Time	Cycles
Initial denaturation	95°C	30 sec	1
Denaturation	95°C	10 sec	40
Annealing/Extension	60°C	30 sec	40
Melting curve	Use the instrument default program	-	-

3. Perform melting-curve analysis after amplification to confirm product specificity. Ramp rates and fluorescence acquisition settings may be adjusted according to the real-time PCR instrument used.