Application

Cloning into vectors, library construction, TA cloning, linker ligation, and re-circlization of linear DNA

Figure 1. Stability test of AccuPower DNA Ligation PreMix at room temperature.

Lane 1 – 12: Lambda DNA / Hind lll fragment (1 µg)
Lane 12 – 24: Lambda DNA / EcoR V fragment (1 µg)
Lane 2, 3, 14, 15: Ligation with AccuPower Ligation PreMix stored at room temperature for 1 month
Lane 5, 6, 17, 18: Ligation with AccuPower Ligation PreMix stored at room temperature for 2 months
Lane 8, 9, 20, 21: Ligation with AccuPower Ligation PreMix stored at room temperature for 3 months
Lane 11, 12, 23, 24: Ligation with AccuPower Ligation PreMix stored at room temperature for 4 months

Figure 2. DNA Ligation efficiency comparison between AccuPower DNA Ligation PreMix and other competitors’ products.

Lane 1, 9: Intact Lambda DNA (1 µg)
Lane 2 – 8: Lambda DNA / Hind lll fragment (1 µg)
Lane 10 – 16: Lambda DNA / EcoR V fragment (1 µg)
Lane 3, 4, 11, 12: Ligation with AccuPower Ligation PreMix
Lane 5, 13: Ligation with T4 DNA Ligase from company N
Lane 6, 14: Ligation with Quick Ligation Kit from company N
Lane 7, 15: Ligation with LigFast Rapid DNA Ligation System from company P
Lane 8, 16: Ligation with Ready-To-Go T4 DNA Ligase from company A

Specifications

5' to 3' exonuclease activity: Yes
3' to 5' exonuclease activity: No
Terminal transferase activity: Yes
Fragment Size: 1 kb

Application

- Gene expression profiling
- Target DNA quantification
- Microbial detection
- SNP analysis
- Viral/bacterial pathogen load determination
- Evaluation of primer pair performance for probe-based real-time PCR

Figure 1. Real-Time PCR Data of AccuPower® DualStar™ qPCR PreMix
AccuPower® DualStar™ qPCR PreMix provides at least 7 orders of magnitude in dynamic range (10⁷ ~ 10¹copies/reaction).
A: Amplification curve. West Nile Virus (WNV) primers and TaqMan-based probe were added into DualStar qPCR Premix. A series of WNV positive control diluents were tested.
B: Standard curve. All data were obtained using Exicycler™ 96 Real-Time Quantitative PCR System (Gentaur).

Figure 2. Data using Various kinds of Real-time PCR Instruments
AccuPower® DualStar™ qPCR PreMix provides at least 7 orders of magnitude in dynamic range (10⁷ ~ 10¹copies/reaction).
A: Amplification curve. and standard curve using ABI 7500 Format Fast Real-time PCR machine (Applied Biosystems). A series of WNV positive control diluents were tested.
B: Amplification curve and standard curve using ABI 7500 Format Real-time PCR machine (Applied Biosystems). 
C: Amplification curve and standard curve using Opticon Real-time PCR machine (MJ Research, currently Bio-Rad).

Figure 3. Comparison of detection sensitivity between AccuPower® DualStar™ qPCR PreMix and other supplier's master mixture
West Nile Virus (WNV) primers and TaqMan-based probe were added into AccuPower® DualStar™ qPCR Premix and other supplier's master mixture. A series of WNV positive control diluents were tested. Reaction mixtures were prepared and qPCR was performed according to each supplier's protocol. All data were obtained using ABI 7500 Format Fast Real-time PCR machine (Applied Biosystems).

Specifications

5' to 3' exonuclease: No
3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment Size: Up to 10 kb

Application

- First-strand synthesis of cDNA from RNA molecules (Reverse Transcription)
- RT-PCR
- Random priming reaction
- Library construction
- Probe labeling
- mRNA 5’end mapping by primer extension analysis
- Real time PCR

Experimental data

Figure 1. Comparison of amplification efficiency between AccuPower RocketScript Cycle RT PreMix and competitors’ RTases
(a) Sensitivity test
Primer set: Human TFRC set
Lane M: 1 kb DNA Ladder
Lane1: 100 ng Human total RNA from HeLa cell
Lane 2: 10 ng Human total RNA from HeLa cell
Lane3: 1 ng Human total RNA from HeLa cell
Lane4: 100 pg Human total RNA from HeLa cell
RT reaction condition is performed according to each manufacturer’s recommendations

(b) Full-Length cDNA synthesis test
RT reactions were performed according to each manufacturer’s recommendation. All cDNAs were amplified with AccuPower Hotstart PCR Premix (K-5050) from Gentaur
Note: Competitor products show inhibition at high input concentrations of total RNA
Lane 1: 1 μg Human total RNA from HeLa cell
Lane 2: 100 ng Human total RNA from HeLa cell
Lane 3: 10 ng Human total RNA from HeLa cell

Figure 2. Complex RNA amplification results of RocketScript Cycle RT PreMix
Each target gene was amplified after performing reverse transcription with AccuPower RocketScript Cycle RT PreMix.
Reverse transcription conditions: Conventional 1 hr incubation at 42°C, 50°C, or 60°C, deactivation at 95°C for 5 min
A: M-MLV Reverse Transcriptase
B: AccuPower RocketScript Cycle RT PreMix with Oligo (dT)20
Lane 1: 100 ng Human total RNA from HeLa cell
Lane 2: 10 ng Human total RNA from HeLa cell

Figure 3. Low copy species enrichment by cycle.
Comparing FTRT (Fixed Temperature Reverse Transcription) to 1 and 10 cycle(s) of CTRT reveal progressive improvement in detected cDNA yield as input copies decrease.

FTRT: 60 min incubation at 50°C followed by 5 min deactivation at 95°C
CTRT: Cycles of 37°C annealing 10 sec, 50°C cDNA synthesis 4 min, 55°C secondary structure melting and cDNA synthesis 
30 sec
Primer set: Human GAPDH
Human Total RNA from HeLa cells
qPCR with AccuPower GreenStar qPCR PreMix (K-6210)

Figure 4. Amplification comparison by cycle 

FTRT (Fixed Temperature Reverse Transcription) conditions:

CTRT (Cyclic Temperature Reverse Transcription) conditions:

Primer set: Human GAPDH and myc set
Lane M: 1 kb DNA Ladder
Lane1: 10 ng Human Total RNA from HeLa Cell
Lane 2: 1 ng Human Total RNA from HeLa Cell
Lane 3: 100 pg Human Total RNA from HeLa Cell
Lane 4: 10 pg Human Total RNA from HeLa Cell

Figure 5. Comparison of amplification results between Gentaur RocketScript^TM Cycle RT PreMix and competitor RTases
All cDNAs were amplified with AccuPower Hotstart PCR Premix (K-5050) from Gentaur
Primer set: human Myc498 bp set
Lane M: 1 kb DNA Ladder
Lane1: 10 ng Human total RNA from HeLa Cell
Lane 2: 1 ng Human total RNA from HeLa Cell
Lane 3: 100 pg Human total RNA from HeLa Cell
Lane 4: 10 pg Human total RNA from HeLa Cell

Specifications

5' to 3' exonuclease: No
3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment Size: Up to 10 kb

Application

- First-strand synthesis of cDNA from RNA molecules (RT)
- RT-PCR
- Random priming reaction
- Library construction
- Probe labeling
- mRNA 5end Mapping by Primer Extension Analysis
- Real time PCR

Figure 1. Sensitivity comparison between AccuPower RocketScript RT PreMix and M-MLV RTase
Sensitivity results of AccuPower RocketScript RT PreMix using GAPDH compared with conventional Reverse transcriptases. 
Each 100 ng – 10 fg of total RNA used for RT and the same amount of RT products used for electrophoresis.
Lane 1: 10 fg Human total RNA from HeLa cell           Lane 2: 100 fg Human total RNA from HeLa cell
Lane 3: 1 pg Human total RNA from HeLa cell            Lane 4: 10 pg Human total RNA from HeLa cell
Lane 5: 100 pg Human total RNA from HeLa cell        Lane 6: 1 ng Human total RNA from HeLa cell
Lane 7: 10 ng Human total RNA from HeLa cell          Lane 8: 100 ng Human total RNA from HeLa cell

Figure 2. Comparison of amplification efficiency between AccuPower RocketScript RT PreMix and competitorsM-MLV RTase.
RocketScript is able to handle a wide range of sample concentrations and transcript lengths so your downstream applications are minimally effected by the reverse transcription step.
Lanes 1-3: 1,000 ng, 100 ng and 10 ng of total RNA from HeLa cells, respectively.

Figure 3. Sensitivity comparison between AccuPower RocketScript RT PreMix and competitor RTases using Real Time PCR
Reverse transcription conditions: conventional 1 hr incubation at 60°C, deactivation at 95°C for 5 min All cDNAs were amplified with AccuPower DualStar™ qPCR PreMix (K-6110) from Gentaur.

Concentration	RocketScript RT PreMix	Supplier Q	Supplier I
10,000	23.91	25.63	24.43
1,000	27.33	28.92	28.03
100	30.62	32.42	30.88
10	33.63	35.43	33.95
Efficiency	104%	103%	108%
Linearity	0.99999	0.9996	0.9995

Specifications

5' to 3' exonuclease: No
3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment size: 9 kb

Application

cDNA synthesis

Figure 1. Reliability and reproducibility test with AccuPower RT PreMix. Each template showcases amplified target genes.

Lane M: 100 bp DNA Ladder (D-1030)
Lane 1-4: Reliability test of each lot with AccuPower RT PreMix

Specifications

3' to 5' exonuclease: No
3’ – A Overhang: No
Fragment size: 9 kb

Application

cDNA synthesis

Figure 1. Reaction condition comparison of Cyclic Reverse Transcription versus conventional reverse transcription PCR. 800 ng of HeLa cell total RNA was reverse transcribed using different reaction conditions and then 800 bp, 1.5 kb, 2.0 kb, and 2.6 kb of human transferrin receptor gene were amplified using AccuPower PCR PreMix.

M: 1 kb DNA Ladder (D-1040)
Lane 1 - 4: Conventional RT with dT₂₀ at 42°C
Lane 5 – 8: Conventional RT with dN₁₂ at 42°C
Lane 9 - 12: Conventional RT with dT₂₀ at 55°C
Lane 13 - 16: Conventional RT with dN₁₂ at 55°C
Lane 17 - 20: Cyclic RT with dT₂₀ by 55°C
Lane 21 - 24: Cyclic RT with dN₁₂ at 55°C

Figure 2. Coxsackie virus A9 (AJ295200) 5’-UTR region amplification with different reverse transcription products. Coxsackie virus RNA was reverse transcribed with different reverse transcription products and then amplified with PCR with AccuPower PCR PreMix. All materials were used in accordance with manufacturer’s instructions.

M: 100 bp DNA Ladder (D-1030)
Lane 1: Reverse transcription with AccuPower RT PreMix and oligo dT primer
Lane 2: Reverse transcription with AccuPower RT PreMix and random primer
Lane 3 – 4, 7 – 8, 11 – 12: Reverse transcription with AccuPower CycleScript RT PreMix and dT₂₀
Lane 5 – 6, 9 – 10, 13 – 14: Reverse transcription with AccuPower CycleScript RT PreMix and dN₁₂
Lane 15 – 16: Reverse transcription with RT product from company C and oligo dT primer
Lane 17 – 18: Reverse transcription with RT product from company C and random primer
Lane 19 – 20: Reverse transcription with RT product from company I and oligo dT primer
Lane 21 – 22: Reverse transcription with RT product from company I and random primer

Figure 3. Long transcript production comparison. Reverse transcription was carried out with various reverse transcription products prior to PCR with AccuPower PCR PreMix.

M: 100 bp DNA Ladder (D-1030)
Gentaur: Gentaur / 1 hour incubation at 55°C
C: Company C / 1 hour incubation at 42°C
I: Company I / 1 hour incubation at 45°C
Lane 1, 3, 5: Product reverse transcribed with oligo dT primer
Lane 2, 4, 6: Product reverse transcribed with random primer

Figure 4. Transferrin receptor gene amplification with various reverse transcription products. Reverse transcription was carried out with four different reverse transcription products according to manufacturer’s instructions and then cDNA was amplified with AccuPower PCR PreMix.

M: 1 kb DNA Ladder (D-1040)
Lane 1 – 5: Reverse transcribed with AccuPower CycleScript RT PreMix (dT₂₀) / 1 hour at 55°C
Lane 6 – 10: Reverse transcribed with AccuPower CycleScript RT PreMix (dN₁₂) / 1 hour at 55°C
Lane 11 – 15: Reverse transcribed with Company C product and oligo dT primer / 1 hour at 42°C
Lane 16 – 20: Reverse transcribed with Company C product and random primer / 1 hour at 42°C
Lane 21 – 25: Reverse transcribed with Company I product and dT primer / 1 hour at 45°C
Lane 26 – 30: Reverse transcribed with Company I product and random primer / 1 hour at 45°C

Figure 5. Reaction temperatures and time comparison. Data A and B demonstrate high thermal stability of AccuPowerCycleScript RT PreMix.

A: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dT₂₀
B: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dN₁₂
Conventional RT #1: 1 hour incubation at 42°C
Cyclic RT #1: Repeat 12 times of 2 minutes at 37°C and 3 minutes at 50°C
Conventional RT #2: 1 hour incubation at 55°C
Cyclic RT #2: Repeat 12 times of 1 minutes at 37°C, 3 minutes at 47°C, and 3 minutes at 55°C
M: 100 bp DNA Ladder (D-1030)
Lane 1, 5, 9, 13, 17, 21, 25, 29: 100 ng of HeLa cell total RNA used for reaction
Lane 2, 6, 10, 14, 18, 22, 26, 20: 10 ng of HeLa cell total RNA used for reaction
Lane 3, 7, 11, 15, 19, 23, 27, 31: 1 ng of HeLa cell total RNA used for reaction
Lane 4, 8, 12, 16, 20, 24, 28, 32: 100 pg of HeLa cell total RNA used for reaction

Figure 6. Data C and D demonstrate superior productivity of AccuPower CycleScript RT PreMix..

C: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dT₂₀
D: Reverse transcription carried out with AccuPower CycleScript RT PreMix and dN₁₂
10 min: 2 times of 2 minutes at 37°C and 3 minutes at 50°C
20 min: 4 times of 2 minutes at 37°C and 3 minutes at 50°C
60 min: 12 times of 2 minutes at 37°C and 3 minutes at 50°C
M: 100 bp DNA Ladder (D-1030)
Lane 1, 5, 9, 13, 17, 21: 100 ng of HeLa cell total RNA used for reaction
Lane 2, 6, 10, 14, 18, 22: 10 ng of HeLa cell total RNA used for reaction
Lane 3, 7, 11, 15, 19, 2: 1 ng of HeLa cell total RNA used for reaction
Lane 4, 8, 12, 16, 20, 24, 28, 32: 100 pg of HeLa cell total RNA used for reaction

Specifications

5' to 3' exonuclease activity: No
3' to 5' exonuclease activity: No
Terminal transferase activity: Yes
Fragment Size: Up to 12 kb

Application

HotStart PCR up to 12 kb
PCR with complex genomic templates/low copy
Templates cDNA
Multiplex PCR reactions

Figure 1. Specificity comparison between AccuPower PCR PreMix and AccuPower HotStart PCR PreMix. 50 ng human genomic DNA was amplified into 2.5 - 3 kb fragments with the two products.

A: AccuPower PCR PreMix
B: AccuPower HotStart PCR PreMix
Lane M: 1 kb DNA Ladder (D-1040)
Lane 1: 2.5 kb product
Lane 2: 3.0 kb product

Figure 2. Specificity comparison of multiplex PCR with various HotStart PCR reagents. Each PCR mixture with 2 ng of human genomic DNA and 2 pairs of primers (p53 + p55 and p55 + p63) was incubated for 2 hours at 37°ree;C prior to performing multiplex PCR.

M: 100 bp DNA Ladder (D-1030)
A: AccuPower HotStart PCR PreMix
B: Control – AccuPower PCR PreMix
C: Company T's antibody mediated HotStart Taq DNA polymerase (0.5 U)
D: Company A’s antibody mediated HotStart Taq DNA polymerase (1.0 U)

Specifications

5' to 3' exonuclease activity: Yes
3' to 5' exonuclease activity: No
3' – A Overhang: Yes
Fragment size: Up to 5 kb from human genomic DNA and 10 kb from Lambda DNA

Application

- High specificity PCR
- High sensitivity PCR
- Low-copy target PCR 
- Multiplex PCR
- cDNA amplification
- TA cloning

Figure 1. Comparison of PCR amplification specificity between AccuPower® PyroHotStart Taq PCR PreMix from Gentaur and other suppliers' Hot start PCR master mix.

PCR reactions were performed according to each supplier's protocol. The PrP gene was amplified from human genomic DNA with two different primer sets, separately. This data shows that AccuPower® PyroHotStart Taq PCR PreMix has higher amplification efficiency and specificity than other suppliers' Hot start PCR master mix.
Lane M: 100 bp DNA Ladder(Gentaur, Cat. No.D-1030)
Lane 1: 100 ng DNA, PrP primer set (500 bp)
Lane 2: 10 ng DNA, PrP primer set (500 bp) 
Lane 3: 100 ng DNA, PrP primer set (705 bp)
Lane 4: 10 ng DNA, PrP primer set (705 bp)

Figure 2. Comparison of PCR amplification specificity between AccuPower PyroHotStart Taq PCR PreMix from Gentaur and other suppliers' Hot start PCR master mix.

The ApoE gene was amplifed from 100 ng of human genomic DNA (The PCR product size is 268bp). This data shows thatAccuPower PyroHotStart Taq PCR PreMix has higher amplificition efficency and specificity than other suppliers' Hot start PCR master mix.
Lane M: 100 bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1: AccuPower PyroHotStart Taq PCR PreMix
Lane 2: Supplier I Hotstart Taq PCR preMix
Lane 3: Supplier S Hotstart Taq PCR masterMix
Lane 4: Supplier T Hotstart Taq PCR masterMix
Lane 5: Supplier Q Hotstart Taq PCR masterMix

Figure 3. AccuPower PyroHotStart Taq PCR PreMix has high amplification efficiency and specificity.

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, 30 cycles of 95°C for 20 sec, 55°C for 40 sec, and 72°C for 1 min, and 72°C for 5 min for final extension.
Lane M: 100bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1: P75/73 primer set (139 bp)
Lane 2: P55/53 primer set (211 bp)
Lane 3: P55/63 primer set (447 bp)
Lane 4: P75/83 primer set (618 bp)
Lane 5: P55/73 primer set (1082 bp)
Lane 6: P65/83 primer set (1296 bp)
Lane 7: P55/83 primer set (1561 bp)

Figure 4. Comparison of PCR amplification between AccuPower PyroHotStart Taq PCR PreMix from Gentaur and other suppliers' Hot start PCR master mix.

Sensitivity test was performed by amplifying the IRGC gene from a serial dilution of human genomic DNA. This data shows that AccuPower PyroHotStart Taq PCR PreMix has higher amplification efficiency than other suppliers' Hot start PCR master mix.
Lane M: 100 bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1: 10 ng human genomic DNA
Lane 2: 1 ng human genomic DNA
Lane 3: 100 pg human genomic DNA
Lane 4: 10 pg human genomic DNA

Figure 5. Comparison of cDNA template amplification between AccuPower PyroHotStart Taq PCR PreMix and other suppliers' Hot start PCR master mix.

cDNA synthesized from 10-fold serial-diluted human total RNA from 10 ng to 10 pg using AccuPower RocketScript™ Cycle RT PreMix (Gentaur, Cat. No. K-2201) was used as a template for PCR amplification. This data shows thatAccuPower® PyroHotStart Taq PCR PreMix has higher amplification efficiency than other suppliers' Hot start PCR master mix.
Lane M: 100 bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1: 10 ng human total cDNA
Lane 2: 1 ng human total cDNA
Lane 3: 100 pg human total cDNA
Lane 4: 10 pg human total cDNA

Specifications

Source: Thermus aquatics
5' to 3' exonuclease activity: Yes
3' to 5' exonuclease activity: No
3' – A Overhang: Yes
Fragment size: Up to 8 kb from human genomic DNA and 10 kb from Lambda DNA

Applications

- Routine PCR
- Primer extension
- TA cloning
- Gene sequencing

Figure 1. Comparison of PCR amplification efficiency between AccuPower Taq PCR PreMix from Gentaur and other suppliers' PCR master mix.

The cycling conditions for AccuPower Taq PCR PreMix were 95°C for 5 min, 35 cycles of 95°C for 20 sec, 55°C for 20 sec, and 72°C for 30 sec. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Target Gene : Human myc.
Lane M : 100 bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1 : 10 ng of human total cDNA
Lane 2 : 1 ng of human total cDNA
Lane 3 : 100 pg of human total cDNA
Lane 4 : 10 pg of human total cDNA

Figure 2. Comparison of PCR amplification efficiency between AccuPower Taq PCR PreMix from Gentaur and other suppliers' PCR master mix.

The cycling conditions for AccuPower Taq PCR PreMix were 95°C for 5 min, 35 cycles of 95°C for 20 sec, 55°C for 20 sec, and 72°C for 30 sec. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Target Gene: IRGC (Immunity-related GTPase family, cinema)
Lane M : 100 bp DNA Ladder (Gentaur, Cat. No. D-1030)
Lane 1 : 10 ng human genomic DNA
Lane 2 : 1 ng human genomic DNA
Lane 3 : 100 pg human genomic DNA
Lane 4 : 10 pg human genomic DNA

Figure 3. Comparison of PCR amplification of long targets between AccuPower Taq PCR PreMix from Gentaur and other suppliers' PCR master mix.

The cycling conditions for AccuPower Taq PCR PreMix were 95°C for 5 min, 30 cycles of 95°C for 20 sec, 65°C for 20 sec, and 68°C for 8 min. PCR reactions using other suppliers' PCR master mix were performed according to each supplier's protocol.
Lane M : 1 kb DNA Ladder (Gentaur, Cat. No. D-1040)
Lane 1 : 3 kb fragment (human tumor protein p53 gene) 
Lane 2 : 4 kb fragment (human beta globin region)
Lane 3 : 4.5 kb fragment (human DNA cross-link repair 1A gene)
Lane 4 : 8 kb fragment (human hemoglobin epsilon 1 gene)