Products
Molecular Biology Reagents | Consumables
Nucleic Acid Amplification
qPCR Series
Color Universal qPCR Master Mix
2×Universal Blue SYBR Green qPCR Master Mix Universal color tracer qPCR Master Mix
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Product Information
Product Name | Cat. No. | Spec. |
2×Universal Blue SYBR Green qPCR Master Mix | G3326-00 | 200 μL |
G3326-01 | 1 mL | |
G3326-05 | 5×1 mL | |
G3326-15 | 15×1 mL |
Product Description/Introduction
2×Universal Blue SYBR Green qPCR Master Mix is a specialized 2× premix for qPCR reactions using the SYBR Green I chimeric fluorescence method. The core component Taq DNA Polymerase is a hot-start DNA polymerase based on the antibody method of confinement, which can effectively inhibit non-specific amplification under low-temperature conditions, and at the same time with the reaction Buffer optimized for qPCR, it is very suitable for the qPCR reaction with high specificity and sensitivity, and it can obtain a good standard curve within a wide quantitative region, and quantify the target gene with accurate, reproducible and highly reliable. At the same time, the product contains a special ROX Passive Reference Dye, which is suitable for use in all qPCR instruments, and there is no need to adjust the ROX concentration on different instruments. Pre-mixed solution is added with blue dye, with the addition of tracer, while supporting the provision of yellow template diluent, when the template is diluted with yellow template diluent and added to the blue reaction pre-mixed solution, the color changes from blue to green, the reaction system can be formulated to play a role in tracing the reaction system process, to prevent the leakage of the addition or wrong addition. The spectra of this blue and yellow dye do not overlap with the qPCR dye and do not affect the results of the reaction.
Storage and Shipping Conditions
Ship with wet ice; store at -20, valid for 12 months.
Product Contents
Component Number | Component | G3326-00 | G3326-01 | G3326-05 | G3326-15 |
G3326-1 | 2x Universal Blue SYBR Green qPCR Master Mix | 200 μL | 1 mL | 5 x 1 mL | 15 x 1 mL |
G3326-2 | 40x Yellow Template Dilution Buffer | 200 μL | 1 mL | 1 mL | 1 mL |
Manual | One copy | ||||
Assay Protocol / Procedures
1. (Optional) Template Dilution
The Yellow Sample Buffer is supplied at a 40X concentration for DNA template dilution, which can accurately determine whether the template has been added to the qPCR reaction solution by the change of liquid color. Taking the 20-μL qPCR reaction system as an example, according to the dilution template amount added into the 20μL qPCR reaction solution, the corresponding dilution method of the original template can be referred to the following table (taking the original template diluted to 100μL as an example):
For example: 2μL diluted template should be added to 20-μL qPCR reaction system. Taking the 100μL Template Dilution system as an example. If the original template volume is 20μL, 25μL 40x Yellow Template Dilution Buffer is added, and then Nuclease-free Water 55μL is added to the total volume of 100μL. The 40x YELLOW Template Dilution Buffer is now 10x. Add 2μL of the diluted template into a 20-μL qPCR reaction system, and the final concentration of 40× YELLOW Template Dilution Buffer is 1x. Or if you do not want to use the template dilution for G3326-2, you can ignore this step.
1. 2. Recommended qPCR reaction system:
Component | 20 μL rxn | 50 μL rxn | Final Concentraction |
2×Universal Blue SYBR Green qPCR Master Mix | 10 μL | 25 μL | 1× |
0.4 μL | 1 μL | 0.2 μM | |
Reverse Primer (10μM)a | 0.4 μL | 1 μL | 0.2 μM |
Templateb | Variable | Variable | as required |
Nuclease-Free Water | Add to 20 μL | Add to 50 μL |
a: Usually, a good amplification effect can be obtained with the final concentration of 0.2 μM. When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1.0 μM.
b: The amount of template added varies depending on the number of copies of the target gene, and the appropriate amount of template addition is studied by gradient dilution. The best addition amount of template DNA in the 20 μL reaction system was less than 100 ng. When the cDNA (RT reaction solution) of RT-PCR reaction was used as template, the addition amount should not exceed 10% of the final qPCR volume.
2. 3. PCR reaction program (can be adjusted appropriately according to the instruments):
B. Three steps method | |||||||||
Step | Cycle number | Temperature | Time | Stage | Step | Cycle number | Temperature | Time | |
Stage 1 | Predegeneration | 1 | 95 | 30 sec | 1 | 95 | 30 sec | ||
Stage 2 | 40 |
95
| 15 sec | Stage 2 | Degeneration | 40 | 95 | 15 sec | |
annealing-extension | 60 | annealing | 55-65 | 10 sec | |||||
extension | 72 | 30 seca | |||||||
Stage 3 | melting curve | 1 | Instrument default Settings | Stage 3 | 1 | ||||
a: If amplification specificity needs to be improved, two-step procedure or annealing temperature can be used; To improve the amplification efficiency, a three-step procedure or extension time can be used.
Note
1. If pipette tracing is not required, template dilution is not performed using the 40×Yellow Template Dilution Buffer.
2. Before using the reagents, please gently invert up and down to mix, do not vortex and oscillate to avoid bubbles.
3. When preparing the reaction solution, keep the reagents on ice.
4. This product contains the fluorescent dye SYBR Green, and strong light should be avoided when preparing PCR reaction solution.
5. Please use new disposable tips for preparation and dispensing of reaction solution to avoid cross contamination between samples as much as possible.
6. Avoid repeated freezing and thawing of Master Mix and try to use it within one month after thawing.
Compatible instruments
ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900 HT Fast, StepOne, StepOne Plus, 7500/7500 Fast, ViiA 7, QuantStudio series, PikoRealTM Cycler;
Stratagene: Mx3000P®, 3005P, 4000;
Bio-Rad: CFX96, CFX384, iCycler iQ, iQ5, MyiQ, MiniOpticon, Opticon®, Opticon 2, Chromo4;
Eppendorf: Realplex 2s, Mastercycler® ep, realplex;
IIIumina: Eco QPCR;
Cepheid: SmartCycler®;
Qiagen Corbett: Rotor-Gene® series;
Roche: LightCycler series;
Takara: Thermal Cycler Dice series;
Analytikjena: qTOWER series;
qTOWER: LineGene series.
Primer design principles
1. The length of amplification product is recommended to be between 80-300 bp;
2. Primer length: 18-25 bp;
3. The content of base G+C in primers should be between 40%-60%;
4. The Tm value difference between forward primers and reverse primers is less than 2, and the Tm value between 58-62 is the best;
5. Randomness of base distribution;
6. Primers had better not contain self-complementary sequences, otherwise they will form a secondary hairpin structure;
7. There should be no more than 4 complementary or homologous bases between two primers, otherwise primer dimer will be formed, especially complementary overlap at the 3' end;
8. The 3' terminal base of the primer is suggested to be G or C;
9. No other non-specific products were found in NCBI comparison results.
Trouble-Shooting
Problem description | Possible reasons | Solutions |
At the end of the reaction, no amplification curve appeared or CT value appeared too late | The template concentration is too low | Repeat the experiment to reduce the template dilution multiple, and start from the highest concentration when the sample concentration is unknown |
Template degradation | The template was prepared again and the experiment was repeated | |
There are PCR inhibitors in the system | Generally, the template is carried in, the dilution ratio of the template is increased or the template with high purity is reprepared and repeated | |
Primers may degrade | Primers that have not been used for a long time should first be tested for integrity by PAGE electrophoresis to rule out the possibility of degradation | |
Low amplification efficiency | Increase the primer concentration, try a three-step amplification procedure, or redesign the primer | |
The amplification product is too long | The amplification product length was controlled in the range of 80-300 bp | |
The blank control shows the signal | Reaction system pollution | Firstly, the blank control water should be replaced. If the same situation still occurs, the primers, aspirators and PCR tubes should be replaced or a new Master Mix should be started. The reaction system is prepared in a super clean table to reduce aerosol pollution |
Non-specific amplification such as primer dimers appears | Generally, it is normal for the amplification products to appear in blank control after 35 cycles, which should be analyzed with the melting curve. Redesign primer, adjust primer concentration or optimize PCR reaction procedure | |
The melting curve has multiple peaks | Primer design is poor | The new primer was re-designed according to the primer design principles |
Primer concentration is too high | Reduce primer concentration appropriately | |
There is genomic contamination in cDNA template | The extracted RNA solution is digested using DNA enzymes, such as dsDNase, to remove genomic contamination, or to design transintron primers | |
Poor reproducibility of experiments | The error of adding sample is large | The use of accurate pipette, with high quality suction head accurate pipette; High dilution template, adding large volume template to reduce sampling error; The reaction volume of qPCR was enlarged |
The template concentration is too low | Repeat the experiment to reduce the dilution times of the template | |
Temperature deviation at different locations of the qPCR instrument | Calibrate the qPCR instrument regularly | |
The amplification curve is not smooth | Fluorescence signal is too weak, produced after system correction | Ensure that the dyes premixed in the Master Mix are not degraded; Replace fluorescent signal to collect better qPCR consumables |
Amplification curve breaks or slips | The template concentration was higher and the baseline endpoint value was greater than the CT value | The baseline endpoint (Ct value -3) was reduced and the data were reanalyzed |
Amplification curves of individual Wells suddenly dropped sharply | There are bubbles in the reaction tube | Ensure that MIX is completely dissolved, and do not swirl and oscillate evenly; After the sample is added, the bubbles are removed by centrifugation with light elastic. The pre-denaturation time was extended to 10 min to remove the bubbles |
For Research Use Only!
