Applications
- Real-Time quantification of DNA and cDNA targets
- Gene expression profiling
- Microbial & Viral pathogen detection
Experimental Data
| Step |
Condition |
Cycle |
| Pre-Denaturation |
95°C, 15min |
1 |
| Denaturation |
95°C, 15sec |
45 |
| Annedling/Extension |
60°C, 30sec |
| Detection(Scan) |
|
| Melting |
65°C~90°C, every 1°C, 1sec |
1 |
| PCR reaction mixture |
Perreaction |
| 2X GreenStar Master Mix |
25ul |
| PCR F-Primer (10 pmole) |
1.5ul |
| PCR R-Primer (10 pmole) |
1.5ul |
| Template |
5ul |
| DEPC-distilledwater. |
A djust to 50ul |
| Copy |
C(t) Value |
| Batch 1 |
Batch 2 |
Batch 3 |
Error Rate |
| NTC |
UD |
UD |
UD |
0.5 C(t) |
| 10 |
36.33 |
36.74 |
35.54 |
| 100 |
32.22 |
31.77 |
31.73 |
| 1000 |
28.51 |
28.7 |
28.04 |
| 10000 |
25.09 |
25.12 |
24.73 |
| 100000 |
21.27 |
21.43 |
21.47 |
| 1000000 |
18.12 |
18.13 |
18.04 |
| 10000000 |
14.99 |
14.43 |
14.45 |
Fig 1. Highly reproducible Ct values
Amplification of an 90-bp target gene was detected using serially diluted LP(Legionella Pneumoniae) genomic DNA (from 106 copies to 101 copies ) with AccuPower® 2X GreenStar qPCR Master mix.
As shown in Fig. Highly reproducible Ct values were achieved within each Lot. set of triplicates
Using a Bioneer Exicycler™ 96
- The annealing temperature can be set to 55~65°C, depending on the primer Tm value.
- The annealing time should be set for 5~20 seconds.
Longer annealing time results in increased efficiency, and a shorter time decreases non-specific amplification.
Fig 2. Comparison of the specificity of the intercalating dye-based real-time PCR products
Amplification of a 90-bp target gene was detected using serially diluted LP (Legionella Pneumoniae) genomic DNA (10n dilution;105~101 copies).
As shown in the Fig, very small amount of primer dimers was observed in AccuPower® 2X GreenStar™ qPCR Master Mix compared with other kits.
| Target Gene |
hGAPDH |
hPTGS2 |
| Sample No. |
1 |
2 |
1 |
2 |
| C(t) Value |
23.73 |
23.46 |
33.98 |
30.52 |
Fig 3. Gene expression analysis
AccuTarget™ Validated Real-Time PCR Primer Library for Human is designed by Bioneer’s bioinformatics tool and targeting for human genome. cDNA was synthesized using Human PTGS2 target primer of those and Human total RNA identically quantified from Hela cell and blood cell with AccuPower® CycleScript RT PreMix(K-2044, Bioneer). Gene analysis was carried out both Hela cell and blood cell by operating Real-Time PCR reaction(95°C 10 min, 1 cycle and 95°C 10 sec, 58°C 25 sec, 72°C 30 sec, 41 cycles) using the cDNA, AccuPower® 2X Greenstar qPCR Master Mix and Exicycler™ 96 Real-Time Thermal Block(Cat. No. A-2060).