AccuPower® Hotstart Pfu PCR PreMix for high specific amplification
AccuPower® HotStart Pfu PCR PreMix is a ready-to-use lyophilized mastermix containing all components for high fidelity PCR. Just addition of primers and template into the tube provides reproducible results. AccuPower® HotStart Pfu PCR PreMix uses a unique enzyme-mediated HotStart PCR method that reduces pre-PCR mis-primings, primer dimers, artifacts, and any other non-specific amplification. Besides AccuPower® HotStart Pfu PCR PreMix provides sensitivity, high specificity and proofreading activity. So you’ll get fewer errors in your PCR product.
Principle
Enzyme-mediated Hotstart PCR method
Phyrophosphate (ppi) has a hjgh binding affinity for Mg and Ppi-Mg complex suppresses a non-specific application before starting the PCR reaction.
Pfu DNA Polymerase is active at the temperature 70°C via ….
Proofreading activity
Polymerase extends complementary strand with primers
If Polymerase adds an incorrect nucliotie, mis-paired nucleotide is removed through proofreading step.
Features and Benefits
High fidelity:
AccuPower® Hotstart Pfu PCR PreMix has the high fidelity which reduces the mispriming during DNA amplification.
Ease-of-Use:
Just add template and primers and start your reaction.
High specificity:
Pyrophosphate (PPi) has a high affinity for Mg2+ and PPi binds to Mg2+ which is essential component for PCR so that DNA polymerase activity is suppressed.. Consequently, PPi-Mg2+ binding prevents non-specific amplification
Reproducibility:
Each product batch is produced under strict quality control processes in large scale batch
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Specifications
5' to 3' exonuclease: No
3' to 5' exonuclease: Yes
3’ – A Overhang: No
Fragment Size: ≥5kb
Application
- Gene cloning with blunt ends
- Site-directed mutagenesis
- High fidelity amplification
- Highly specific PCR
- cDNA amplification
Experimental Data
Figure 1. Comparison of PCR amplification efficiency between AccuPower® Hotstart Pfu PCR PreMix of Bioneer and other suppliers' PCR master mix
Target: Human insulin receptor gene. The cycling conditions for AccuPower® Hotstart Pfu PCR PreMix were 95°C for 5 min, 30 cycles of 95°C for 30 sec, 55°C for 1 min and 72°C for 5min. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Lane M : 100 bp DNA Ladder(Bioneer, Cat. No. D-1030)
Lane 1 : 10 ng of human genomic DNA
Lane 2 : 1 ng of human genomic DNA
Lane 3 : 100 pg of human genomic DNA
Lane 4 : 10 pg of human genomic DNA
Figure 2. AccuPower® Hotstart Pfu PCR PreMix shows enhanced specificity compared to competitors.
Specificity test was performed using 7 different sets of primers targeting the P53 gene. 10 ng of human genomic DNA was used for each PCR reaction. The cycling conditions were 95°C for 5min, 32 cycles of 95°C for 30 sec, 62°C for 40 sec, and 72°C for 1 min 30sec, and 72°C for 5 min for final extension.
Lane 1: P75/73 primer set (139bp) Lane 2: P55/53 primer set (211bp) Lane 3: P55/63 primer set (447bp)
Lane 4: P75/83 primer set (618bp) Lane 5: P55/73 primer set1082bp) Lane 6: P65/83 primer set (1296bp)
Lane 7: P55/83 primer set (1561bp) Lane M: 100bp DNA Ladder (Bioneer, D-1030)
Figure 3. Comparison of PCR amplification specificity between AccuPower® Hotstart Pfu PCR PreMix of Bioneer and other suppliers' Pfu DNA polymerase.
Three different genes with two concentrations (100ng and 10ng) were amplified under the same conditions. The cycling conditions were 95°C for 5min, 35 cycles of 95°C for 30 sec, 62°C for 30 sec, and 72°C for 1 min 30 sec, and 72°C for 5 min for final extension.This data shows that AccuPower® Hotstart Pfu PCR PreMix has higher amplificition efficency and specificity than other suppliers' Pfu DNA polymerase.
Lane M: 100 bp DNA Ladder(Bioneer, Cat. No.D-1030)
Lane 1: 100 ng DNA, ApoE primer set (268 bp)
Lane 2: 10 ng DNA, ApoE primer set (268 bp)
Lane 3: 100 ng DNA, PrP primer set (500 bp)
Lane 4: 10 ng DNA, PrP primer set (500 bp)
Lane 5: 100 ng DNA, PrP primer set (705 bp)
Lane 6: 10 ng DNA, PrP primer set (705 bp)
Figure 4. AccuPower® Hotstart Pfu PCR PreMix has high amplification efficiency.
The cycling conditions were 95°C for 5min, 35 cycles of 95°C for 20 sec, 65°C for 20 sec, and 68°C for 15 min , and 68°C for 5 min for final extension.
Lane M: 1 kb DNA Ladder (D-1040; Bioneer)
Lane 1: 2 kb fragment (Human tumor protein p53 gene)
Lane 2: 2.5 kb fragment (Human tumor protein p53 gene)
Lane 3: 3 kb fragment (Human tumor protein p53 gene)
Lane 4: 4.5 kb fragment (Human DNA cross-link repair 1A gene)
Lane 5: 5kb fragment (Human mitocodria gene)
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Manual
• AccuPower® Hotstart Pfu PCR PreMix
MSDS
• MSDS_AccuPower® Hotstart Pfu PCR PreMix
Brochure
• AccuPower® Hotstart Pfu PCR PreMix - 2012 Brochure
Quality Assurance
Bioneer is the holder of Quality Management System Certificates for the following standards.
• ISO 9001:2008 - certificate
• EN ISO 13485:2003 / AC:2009 - certificate
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