2×Universal Blue SYBR Green qPCR Master Mix (with UDG)

Product Information

Product Description

This product is a specialized 2× premix for qPCR reactions using the SYBR Green I chimeric fluorescence method. The use of antibody-enclosed Taq DNA Polymerase in combination with UDG (Uracil-DNA Glycosylase) and dUTP effectively reduces the formation of primer dimers, enhances specificity, and improves sensitivity on the one hand, and prevents cross-contamination caused by PCR amplification products on the other hand, resulting in a more accurate quantification of the target gene. Meanwhile, this product contains special ROX Passive Reference Dye and blue visualization spiking tracer dye, which can be applied to all qPCR instruments, without the need to adjust the ROX concentration on different instruments. The yellow template diluent is also provided. When the template is diluted with the yellow template diluent and added to the blue reaction premix, the color changes from blue to green, which can play a tracer role in the process of preparing the reaction system, preventing the omission 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°C away from light, valid for 12 months.

Product Contents

Assay Protocol

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.

Note: If the concentration of the template is too low, do not need to dilute, or do not want to use diluted template with 40x YELLOW Template Dilution Buffer, you can omit this step.

1. 2. Recommended qPCR reaction system: 

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):

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 pipetting tracking is not required, dilute template with the 40×Yellow Template Dilution Buffer can be omitted.

2. Please mix the reagents before use by gently inverting them up and down. Do not vortex and oscillate to avoid bubbles.

3. Reagents should be placed on ice when preparing reaction mixes.

4. Strong light should be avoided when preparing PCR reaction mixes due to fluorescent dye SYBR Green.

5. New disposable tips should be used for preparation of reaction mixes to avoid cross contamination

6. Avoid freeze-thawing cycles of the Master Mix, and try to use it up within a month after thawing.

Compatible Instruments

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne Plus, 7500/7500 Fast, ViiA 7, QuantStudio Series, PikoRealTM Cycler.

Stratagene: Mx3000P®, 3005P,

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;

Hangzhou Bori: LineGene department;

Primer Design Principles

1. The length of the amplified product is recommended to be between 80-300 bp;

2. Primer length: 18-25 bp;

3. Keep the G＋C content in the 40%-60% range.

4. It is preferable that the Tm values of the forward and reverse primers do not differ by more than 2°C, and it is better to control the Tm value at 58-62°C;

5. Randomness of base distribution; 

6. There should be no more than four complementary or homologous bases between the two primers, otherwise a primer dimer will be formed, and complementary overlap at the 3' end should be avoided in particular;

7. It is recommended that the 3' end base of the primer be G or C;

8. No non-specific products appear on NCBI blast.

Trouble-Shooting

For Research Use Only!

https://www.servicebio.com/goodsdetail?id=13304