2 × SYBR Green qPCR Master Mix (None ROX)
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Product Information
Product Name | Cat. No. | Spec. |
2×SYBR Green qPCR Master Mix (None ROX) | G3320-01 | 1 mL |
G3320-05 | 5×1 mL | |
G3320-15 | 15×1 mL |
Product Description/Introduction
2×SYBR Green qPCR Master Mix (None ROX) is a special 2× premix for qPCR reaction using SYBR Green I chimeric fluorescence method, which contains all qPCR components except primers and DNA templates, which can reduce the operation steps, shorten the time of adding samples, and reduce the chance of contamination. The core component is genetically engineered hot-start Taq DNA Polymerase, which effectively seals off DNA polymerase activity and prevents non-specific amplification at low temperatures by efficiently
combining monoclonal antibody and Taq DNA Polymerase, with many advantages such as high specificity and high sensitivity, and is coupled with a reaction buffer optimized for qPCR. It is very suitable for high specificity and high sensitivity qPCR reaction. This product is a 2× premixed reagent containing the optimal concentration of SYBR Green I for qPCR reaction, which can obtain a good standard curve in a wide quantification area, accurate quantification of target genes, good reproducibility and high confidence.
Storage and Shipping Conditions
Ship with wet ice. Store at -20°C without light, valid for 12 months. Avoid freeze-thaw cycles. After thawing, it can be stably stored at 4 for one month without light.
Product Contents
Component | G3320-01 | G3320-05 | G3320-15 |
2×SYBR Green qPCR Master Mix (None ROX) | 1 mL | 5×1 mL | 15×1 mL |
Manual | One copy | ||
Assay Protocol / Procedures
Before starting
1. Real Time PCR amplification apparatus;
2. Special reaction tube or reaction plate for experiment;
3. PCR primers (reference primer design principles);
4. Micropipette and autoclaving-tips;
Procedures
1. Recommend the qPCR reaction system:
Component | 20 μL rxn | 50 μL rxn | Final Concentration |
2×SYBR Green qPCR Master Mix (None ROX) | 10 μL | 25 μL | 1× |
Forward Primer (10 μM)a | 0.4 μL | 1 μL | 0.2 μM |
Reverse Primer (10 μM)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. PCR reaction program (can be adjusted appropriately according to the instrument)
A. Two-step process * | B. Three-step process* | ||||||||
Stage | Step | Cycles | Temperature | Time | Stage | Step | Cycles | Temperature | Time |
Stage 1 | Predegeneration | 1 | 95 | 30 sec | Stage 1 | Predegeneration | 1 | 95 | 30 sec |
Stage 2 | Degeneration | 40 | 15 sec | Stage 2 | Degeneration | 40 | 95 | 15 sec | |
Annealing-Extension | 60 | 30 seca | Annealing | 55-65 | 10 sec | ||||
Extension | 72 | 30 seca | |||||||
Stage 3 | Melting curve | 1 | Instrument default Settings | Stage 3 | Melting curve | 1 | Instrument default Settings | ||
*: 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.
a: For fluorescence signal collection, please set the experimental procedure according to the instruction manual of the instrument.
Note
1. Mixed gently upside down before use. Do not swirl and shake to avoid bubbles. Reagents are mixed well before use.
2. Reagents should be placed on ice when preparing reaction solution.
3. The product contains fluorescent dye SYBR Green, so strong light should be avoided when preparing PCR reaction solution.
4. Please using new disposable tips for the preparation and packaging of the reaction solution to avoid contamination between samples.
5. Avoid repeated freeze-thawing of Master Mix and try to use it within one month after thawing.
Compatible instruments
Brand of PCR machine | G3320 (None ROX) | G3321 (Low ROX) | (High ROX) |
ABI Thermo life | PikoRealTM Cycler | 7500/7500 Fast， ViiA 7 QuantStudio series | 5700/7000/7300/7700/7900/ 7900HT/7900 HT Fast，StepOne，StepOne Plus |
Stratagene | Mx3000P®/3005P/4000 | ||
Bio-Rad | All series | ||
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
Problems | Possible cases | Solutions |
At the end of the reaction, no amplification curve appeared or CT value appeared too late | Template concentration too low | Reduce the number of template dilutions to repeat the experiment, starting with the highest concentration if the sample concentration is unknown. |
Degradation of primers | Reprepare the template and repeat the experiment. | |
PCR inhibitors present in the reaction mixture. | Generally template carryover, increase template dilution or re-prepare template with high purity to repeat the experiment. | |
Possible degradation of primers | Primers that have not been used for a long period of time should first be tested for integrity by PAGE electrophoresis to rule out their degradation. | |
Low amplification efficiency | Increase the primer concentration, try a three-step amplification program, or redesign the primers. | |
Amplification products too long | Amplification product length was controlled at 80-300 bp. | |
Amplification signal in non-template control | Reaction system contamination | · First change the water of the blank control, if the same thing still happens, continue to change the primers, tips, PCR tubes or enable a new Master Mix; The reaction system is prepared in an ultra-clean bench to minimize aerosol contamination. |
Non-specific amplification such as primer dimers | Generally, it is normal for amplification products to appear in the blank control after 35 cycles, which should be analyzed together with the melting curve; redesign the primers, adjust the primer concentration or optimize the PCR reaction procedure. | |
The melting curve has multiple peaks | Primer design is suboptimal | New primers were redesigned according to primer design principles. |
Primer concentration is too high | Reduce primer concentration appropriately | |
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 | Reduce the template dilution times to repeat the experiment. | |
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!
