2 × Fast SYBR Green qPCR Master Mix (Low ROX, 5mL)

 

 

a. Usually, a good amplification effect can be obtained with the final concentration of 0.2 μ M . When the reaction performance is poor, the primer concentration can be adjusted in the range of 0.2-1.0 μ M .  

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 μ L 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  . 

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 com plex 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 s ec .
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; Orescence signal collection, please set the experimental procedure according to the instruction manual of the instrument.

Note

• Mixed gently upside down before use. Do not swirl and shake to avoid bubbles.
• Reagents should be placed on ice when preparing reaction solution.
• The product contains fluorescent dye SYBR Green, so strong light should be avoided when preparing PCR reaction solution.
• Please use new disposable head for the preparation and packaging of the reaction solution to avoid contamination between samples.
• Avoid repeated freeze-thaw Master Mix and try to use it within one month after thawing.

Compatibility

Brand of PCR machine	G3323 (None ROX)
ABI Thermo life	7500/7500 Fast， ViiA 7™ QuantStudio™ series
Stratagene	Mx3000P®/3005P™/4000™

 

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