SYBR Green Fast qPCR Premix HYA411
Real-time Quantitative PCR is a technique using DNA double-stranded dyes, usually SYBR® Green I, to quantitative detect the initial amount of DNA by amplification. SYBR Green Fast qPCR Premix is a fluorescence reagent for qPCR reactions using the SYBR® Green I. This product provides real-time data on DNA amplification during PCR by performing quantitative fluorescence signal detection on SYBR®/FAM channels. This product uses the antibody-based hot-start Taq enzyme for amplification, which greatly improves the specificity of the product while ensuring the amplification effect. At the same time, by optimizing the qPCR Mix Buffer system, the product is suitable for multiple species, providing a powerful tool for multidisciplinary experimental needs. This product is a 2X pre-mixed enzyme, contains all the components required for qPCR except primers and templates, providing great convenience for experimental manipulation.
Components
|
Components |
1 mL |
5 mL |
25 mL |
|
SYBR Green Fast qPCR Premix* |
1mL |
1mL×5 |
1mL×25 |
|
50X ROX Reference Dye I |
40 μL |
200 μL |
200 μL×5 |
|
50X ROX Reference Dye II |
40 μL |
200 μL |
200 μL×5 |
*Including Hot Start Taq DNA polymerase, Mg2+, dNTPs, SYBR® Green I, etc
Storage
This product should be stored at -20℃ for long-term storage and should be protected from light.
Materials Required
1. PCR tubes and other related materials.
2. qPCR specific primers and DNA templates.
3. qPCR 96-well plate and sealing membrane (adhesive film).
Notes
1. SYBR Green Fast qPCR Premixmust be completely thawed before use. Protect from direct light exposure and store in darkness.
2. SYBR Green Fast qPCR Premixcontains glycerol. Gently mix before use to avoid bubble formation; vortex and centrifuge before use. Immediately return to -20°C storage after use.
3. This product contains DNA polymerase. Maintain on ice throughout use. For short-term repeated usage within a day, temporary storage at 4°C is permitted. Minimize freeze-thaw cycles.
Instruments
1. Experimental Preparation
1. It is recommended to choose the amplification product length within the range of 70-200 bp.
2. It is recommended to take a reaction volume of 20 μL, add 1 pg-50 ng of DNA as a template, and set NTC (No Template Control).
3. To ensure experimental accuracy, perform triplicate technical replicates for all samples and controls.
4. The ROX Reference Dye is different for different instruments. For adding or not, please refer to the table below.
|
Rox types |
qPCR Machines |
|
No ROX |
Bio-Rad iCycler serious, Roche Light Cycler serious, Qiagen/Corbett serious and others |
|
50X ROX Reference Dye I |
ABI 7000/7300/7700/7900, ABI StepOne/StepOnePlus, Eppendorf and others |
|
50X ROX Reference Dye II |
ABI 7500, ABI ViiATM7, ABI QuantaStudio serious, Stratagene serious, Corbett Rotor Gene 3000 and others |
2. qPCR Reactionsystem
|
Components |
Volume |
|
SYBR Green Fast qPCR Premix |
10uL |
|
Forward Primer (10 µM)** |
0.4 uL |
|
Reverse Primer(10 µM)** |
0.4 uL |
|
gDNA or cDNA* |
2uL |
|
ROX I / II(ifrequired, select according to instrument model) |
0.4 uL |
|
ddH2O |
Up to 20 uL |
* Typically, the final concentration of the primer is 0.2 μM，and good results can be obtained , and the final concentration of 0.1-1.0 μM can be used as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the concentration of primers can be reduced and the reaction system can be optimized.
** Using 10 pg-10 ng genomic DNA or 10 pg-100 ng cDNA as the template reference quantity, gradient dilution can be performed on the template to determine the optimal template usage due to the different copy numbers of the target genes contained in the templates of different species. In addition, when using cDNA (RT reaction solution) from the two-step RT qPCR reaction as a template, the addition amount should not exceed 10% of the qPCR reaction system.
3. qPCR ReactionProgram
|
Step |
Temperature |
Time |
Cycles |
|
Pre Denaturation |
95 ℃ |
3 min |
1 |
|
Denaturation |
95 ℃ |
5 s |
40-45 |
|
Annealing and Extension |
60℃ |
30-34 s* |
|
|
Melt Curve |
Instrument default |
/ |
1 |
* Confirm there is a signal collection step after each extending step. The extending time is varied according to different machines: 30 s for StepOne Plus, 31 s for 7300 and 34 s for 7500. If not otherwise specified, default to 30 s.