Sanshibio 2× SYBR Green qPCR Mix

Item No： EH003          Specifiation：1mL          Storage: Store at -20°C

Product introduction：

• Specialized reagent for Real-Time PCR utilizing the SYBR Green I dye-based fluorescence method.
• Contains an antibody-blocked hot-start enzyme to suppress nonspecific amplification caused by primer dimerization or nonspecific annealing of primers under low-temperature conditions.
• Enhances the specificity of the amplification reaction.
• Features high amplification efficiency, strong detection specificity and sensitivity, good stability, and easy operational use.
• Generates a robust standard curve within a broad quantitative range, enabling accurate quantification.
• Compatible with a variety of fluorescence quantitative PCR instruments, including those from Applied Biosystems, Eppendorf, Bio-Rad, Roche, and other domestic brands.

Product contents：

Note：

• ROX Reference Dye corrects inter-well fluorescence signal errors in certain Real-Time PCR amplification instruments.
• ROX Reference Dye I is suitable for ABI PRISM 7000/7700/7300/7900HT and Step One Plus Real-Time PCR System.
• ROX Reference Dye II is compatible with 7500 Real-Time PCR System, 7500 Fast Real-Time PCR System, Stratagene Mx3000P, Mx3005P, and Mx4000.
• Both ROX Reference Dye I and II should be used at a final concentration of 1× in reactions.
• Instruments like LightCycler, Thermal Cycler Dice Real Time System II, and Smart Cycler System do not require the use of ROX Reference Dye.

Storage:

Store at -20°C, with a minimum shelf life of 12 months.

Activity Definition:

Using activated mahi-mahi sperm DNA as template/primer, the activity is defined as 1 unit (U) of acid-insoluble material incorporated, by taking up 10 nmol of nucleotides within 30 minutes at 74°C.

Quality Control:

This product has undergone quality testing and is free from deoxyribonuclease endonuclease activity, deoxyribonuclease exonuclease activity, and ribonuclease contamination. Host genomic DNA residual content is below 10 copies.

Product Uses:

Real-time fluorescence Quantitative qPCR (Dye Method) amplifies DNA or cDNA; Absolute Quantitative qPCR; Relative Quantitative qPCR.

Instructions for Use:

• Allow the required reagents to equilibrate to room temperature until completely dissolved, gently mix thoroughly (do not vortex), use after brief centrifugation to avoid excessive bubble formation and repeated freeze-thaw cycles. If used frequently, store at 4°C. Prepare the PCR reaction mix according to the components pressed down (prepare the reaction mix on an ice box):

Note：The amounts of each component can be adjusted according to actual needs. The addition of ROX Reference Dye should be determined by users based on the specific model being used.

• In general, a two-step method can be used for the reaction. If amplification is not satisfactory with the two-step method, a three-step method can be employed to set up the PCR reaction program.

Note： The reaction conditions can be adjusted and optimized according to actual needs. The melting curve program should be selected and adapted based on the amplification instrument’s program being used.

• After the reaction is complete, analyze the amplification curve and melting curve results. Refer to the Real-Time PCR instrument’s user manual for detailed analysis methods.

Precautions:

1. Please select appropriate annealing (extension) temperatures based on primer design, with the primer Tm typically around 60°C. For primers with lower annealing temperatures, a three-step method is recommended. The final concentration of used primers can be adjusted within the range of 0.2-1.0μM. The DNA template concentration can be adjusted according to its concentration.
2. SYBR Green I dye is a non-specific dye that emits fluorescence upon binding to double-stranded DNA (dsDNA). However, it is easily degraded under strong light. Therefore, when designing primers, it is important to avoid primer dimer formation as much as possible. During usage, prolonged exposure to strong light should be avoided to enhance result accuracy, sensitivity, and specificity.
3. Increasing the magnesium ion concentration can reduce the inhibitory effect of SYBR Green I on PCR reactions. When optimizing fluorescence PCR reactions using this product, it is advisable to slightly increase the magnesium ion concentration (0.5-3.0mM higher than standard PCR reactions).
4. Use dedicated areas and pipettes before and after amplification, wear gloves, and change them frequently. After completing the PCR reaction, do not open the reaction tubes to minimize contamination of the testing environment with PCR products.