Example 1. Amplification of the 100~500bp region

Using artificial gene as template and forward primer as common, reverse primer was designed so that the amplification product lengths were 100bp, 200bp, 300bp, 400bp and 500bp. We used these primers to amplify 107 to 10 copies using THUNDERBIRD™ Next
SYBR^® qPCR Mix and other companies to compare the PCR efficiency for each targets. As a result, the amplification efficiency may decrease or be undetectable as the target length increases in other companies, but stable PCR efficiency was achieved with THUNDERBIRD™ Next SYBR^® qPCR Mix.

Example 2.Amplification from 1 copy gene

If one copy target can be amplified, the number of detected copies would be equivalent to the expected number of detected copies from the Poisson distribution. When one copy is added, the theoretical value from the Poisson distribution is 37% of the probability of having 0 copies and 63% of the probability of having one or more copies.
THUNDERBIRD™ Next SYBR^® qPCR Mix was used to detect 96 samples using Salmonella genome diluted to one copy as a template.
The results showed that 38.5% of the samples were undetected and 61.5% were detected, which is equivalent to the expected number of samples from the Poisson distribution, suggesting that one copy equivalent can be detected.

Example 3.Amplification of 65bp domain of G3PDH

cDNA from total RNA of Hela cells synthesized by reversetranscription reagents (Code No. FSQ-101) was used to amplify a 65bp G3PDH gene at 5-fold dilutions of cDNA. As a result, nonspecific amplification occurred in the low copy range in other companies, but nonspecific amplification was not observed by using THUNDERBIRD™ Next SYBR^® qPCR Mix, allowing accurate quantification to the low copy range.

Example 4.Comparison of normal cycle and high-speed cycle

Using cDNA from Hela cells total RNA synthesized by reverse-transcription reagents (Code No. FSQ-101), amplification of β-actin gene (316bp) was amplified by "normal cycle with 30sec. extension " and "high-speed cycle with 10sec. extension". As a result, other company products that recommend normal cycling could not be amplified efficiently by high-speed cycling, but THUNDERBIRD™ Next SYBR^® qPCR Mix could be amplified efficiently by high-speed cycling.

Example 5. Stability test of the prepared PCR reaction solution

Primers and template (cDNA from Hela cells total RNA) were mixed into the PCR-reaction solution, and amplifications of the targets were performed immediately or after standing for 48 hours at a light-shielding room temperature. As a result, Ct values decreased after 48 hours in our conventional kit and other products. But in THUNDERBIRD™ Next SYBR^® qPCR Mix, Ct values remained stable even after 48 hours.

Example 6. Prevention of false positives

To confirm UNG treatment for preventing carry-over contamination. PCR products containing dUTP (104 copies) were used as template, THUNDERBIRD™ Next SYBR^® qPCR Mix and Uracil-DNA Glycosylase(UNG), Heat-labile(Code No. UNG-101) were added, and amplification of the same target was performed by real-time PCR. As a result, we were able to confirm that the first PCR products were degraded by UNG treatment completely.

Example 1. Amplification of the 100~500bp region

Using artificial gene as template and forward primer as common, reverse primer was designed so that the amplification product lengths were 100bp, 200bp, 300bp, 400bp and 500bp. We used these primers to amplify 107 to 10 copies using THUNDERBIRD™ Next
SYBR^® qPCR Mix and other companies to compare the PCR efficiency for each targets. As a result, the amplification efficiency may decrease or be undetectable as the target length increases in other companies, but stable PCR efficiency was achieved with THUNDERBIRD™ Next SYBR^® qPCR Mix.

Example 2.Amplification from 1 copy gene

If one copy target can be amplified, the number of detected copies would be equivalent to the expected number of detected copies from the Poisson distribution. When one copy is added, the theoretical value from the Poisson distribution is 37% of the probability of having 0 copies and 63% of the probability of having one or more copies.
THUNDERBIRD™ Next SYBR^® qPCR Mix was used to detect 96 samples using Salmonella genome diluted to one copy as a template.
The results showed that 38.5% of the samples were undetected and 61.5% were detected, which is equivalent to the expected number of samples from the Poisson distribution, suggesting that one copy equivalent can be detected.

Example 3.Amplification of 65bp domain of G3PDH

cDNA from total RNA of Hela cells synthesized by reversetranscription reagents (Code No. FSQ-101) was used to amplify a 65bp G3PDH gene at 5-fold dilutions of cDNA. As a result, nonspecific amplification occurred in the low copy range in other companies, but nonspecific amplification was not observed by using THUNDERBIRD™ Next SYBR^® qPCR Mix, allowing accurate quantification to the low copy range.

Example 4.Comparison of normal cycle and high-speed cycle

Using cDNA from Hela cells total RNA synthesized by reverse-transcription reagents (Code No. FSQ-101), amplification of β-actin gene (316bp) was amplified by "normal cycle with 30sec. extension " and "high-speed cycle with 10sec. extension". As a result, other company products that recommend normal cycling could not be amplified efficiently by high-speed cycling, but THUNDERBIRD™ Next SYBR^® qPCR Mix could be amplified efficiently by high-speed cycling.

Example 5. Stability test of the prepared PCR reaction solution

Primers and template (cDNA from Hela cells total RNA) were mixed into the PCR-reaction solution, and amplifications of the targets were performed immediately or after standing for 48 hours at a light-shielding room temperature. As a result, Ct values decreased after 48 hours in our conventional kit and other products. But in THUNDERBIRD™ Next SYBR^® qPCR Mix, Ct values remained stable even after 48 hours.

Example 6. Prevention of false positives

To confirm UNG treatment for preventing carry-over contamination. PCR products containing dUTP (104 copies) were used as template, THUNDERBIRD™ Next SYBR^® qPCR Mix and Uracil-DNA Glycosylase(UNG), Heat-labile(Code No. UNG-101) were added, and amplification of the same target was performed by real-time PCR. As a result, we were able to confirm that the first PCR products were degraded by UNG treatment completely.