9. 2 × Fast SYBR Green qPCR Master Mix (High ROX), 1ml $59,

Product Information

Description/ Introduction

This product is a special 2 × premixed solution for qPCR reaction using SYBR Green I chimeric fluorescence method. It contains all qPCR components except primers and DNA templates, which can reduce the operation steps, shorten the sample addition time and decrease the probability of contamination. The core component is the genetically engineered heat-activated Taq DNA Polymerase, which effectively blocks DNA Polymerase activity and prevents nonspecific amplification at low temperatures by efficiently binding monoclonal antibodies to the Taq DNA Polymerase. The heat-activated Taq DNA Polymerase has many advantages such as high specificity and detection sensitivity. With the reaction buffer optimized for qPCR, it is very suitable for high-specificity and high-sensitivity qPCR reactions. 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 quantitative area. The quantification of target genes is accurately, reproducible and reliable. At last, the qPCR reaction can be completed in 30 minutes at the fastest.

Storage and Handling Conditions

Transport with wet ice. Store at -20°C without light, valid for 12 months. Avoid freeze/thaw cycle. After thawing, it can be stably stored at 4℃ for one month without light.

Component

Assay Protocol / Procedures

Preparation before experiment

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 pipette head (autoclaving);

Procedures

1.Recommend the 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 addition varies with the copy number of the target gene in the template solution, and the appropriate amount of template addition is discussed 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 total volume of PCR reaction solution.

2. PCR reaction procedure (can be adjusted according to the instruments):

It is recommended to use the fast procedure of fluorescent quantitative PCR instrument of the corresponding brand preferentially, and it is compatible with standard amplification procedure. The fast procedure is suitable for most genes, and the standard procedure can be tried for some genes with complex secondary structure.

a: The genome template can prolong the pre-degeneration time by 2-3 min;

b: Cycle stage: the denaturation time of standard procedure is 10 sec. If the amplified fragment is less than 200 bp, the shortest option for fast procedure is 3 sec.

c: Cycle stage: standard procedure annealing/elongation time is selected for 30 sec; If the amplified fragment is less than 200 bp, the shortest procedure could be selected as 10 sec. Over 200 bp, the recommended choice is 30 sec; 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.

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 head for the preparation and packaging of the reaction solution to avoid contamination between samples.

5.Avoid repeated freeze-thaw Master Mix and try to use it within one month after thawing.

Compatibility

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

For Research Use Only!

Ver. No.: V1.0-202102