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 GENTAUR Europe BVBAVoortstraat 49, 1910 Kampenhout BELGIUM Tel 0032 16 58 90 45 Fax 0032 16 50 90 45 This email address is being protected from spambots. You need JavaScript enabled to view it.">This email address is being protected from spambots. You need JavaScript enabled to view it. |
GENTAUR BULGARIA
53 Iskar Str. 1191 Kokalyane, Sofia
Tel 0035924682280
Fax 0035929830072
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GENTAUR France SARL
9, rue Lagrange, 75005 Paris
Tel 01 43 25 01 50
Fax 01 43 25 01 60
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GmbH Marienbongard 20
52062 Aachen Deutschland
Tel (+49) 0241 56 00 99 68
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GENTAUR Ltd.
Howard Frank Turnberry House
1404-1410 High Road
Whetstone London N20 9BH
Tel 020 3393 8531
Fax 020 8445 9411
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GENTAUR Poland Sp. z o.o.
ul. Grunwaldzka 88/A m.2
81-771 Sopot, Poland
Tel 058 710 33 44
Fax 058 710 33 48
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GENTAUR Nederland BV
Kuiper 1
5521 DG Eersel Nederland
Tel 0208-080893
Fax 0497-517897
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GENTAUR SRL IVA IT03841300167
Piazza Giacomo Matteotti, 6, 24122 Bergamo
Tel 02 36 00 65 93
Fax 02 36 00 65 94
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GENTAUR Spain
Tel 0911876558
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Genprice Inc, Logistics
547, Yurok Circle
San Jose, CA 95123
Phone/Fax:
(408) 780-0908
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GENPRICE Inc. invoicing/ accounting:
6017 Snell Ave, Suite 357
San Jose, CA. 96123
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Macedonia,
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Croatia:
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Fax 0035929830072
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GENTAUR Romania
Tel 0035929830070
Fax 0035929830072
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Tel 00302111768494
Fax 0032 16 50 90 45
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Luxembourg +35220880274
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AccuPower CycleScript RT PreMix
AccuPower CycleScript RT (available with: dT20, dN12, or dN6 primers) PreMix is an easy to resuspend lyophilized PCR Master Mix of CycleScript Reverse Transcriptase, a primer, and all of the other components for cDNA synthesis conveniently packaged in individual tubes. Simply add template RNA, and water. Then, the reverse transcription is performed by either a Cyclic RT reaction (patent pending) or conventional reverse transcription PCR. The use of Cyclic RT produces cDNA amplification and better results compared to conventional reverse transcription PCR – especially for rare transcripts. The resulting cDNA can be used in a variety of applications such as reverse transcription PCR (RT-PCR). The AccuPower CycleScript RT (dT20, dN12, or dN6) PreMix series is stable for 2 years at - 20°C due to a patented stabilizer.
AccuPower CycleScript RT was developed for both a conventional reverse transcription PCR at a fixed temperature at 42°C as well as Cyclic Reverse Transcription that is carried out like a PCR (please see the following comparison table). Gentaur’s Cyclic RT is an innovative technology to synthesize more homogeneous cDNA in less time compared to the conventional reverse transcription.
Conventional Reverse Transcription
| Step 1: | RNA denaturation at 65°C for 10 minutes |
| Step 2: | cDNA synthesis at a temperature between 37°C ~ 55°C for 15 ~ 60 minutes |
Cyclic Reverse Transcription
| Step 1: | Primer annealing at a temperature between 15°C and 40°C for 30 seconds |
| Step 2: | cDNA synthesis at a temperature between 42°C and 48°C for 4 minutes |
| Step 3 (optional): | Denaturation of the secondary structure of the RNA template and cDNA synthesis at a temperature between 50°C and 55°C for 30 seconds |
Features and Benefits
| Convenient lyophilized RT premix: | Simply add your purified RNA and start your reaction. Enzyme, dNTPs, reaction buffer, and oligo (dT)20 are provided. |
| Broad range of working temperatures: | For RNAs that are G:C rich or significant secondary structure |
| Novel Cyclic Reverse Transcription: | Ensures that you isolate even the rarest transcripts |
| High yield of cDNA: | For genes up to 9 kb within 10 minutes |
| Stable for 2 years at -20°C: | Long life |
| RNase, DNase and Proteinase-free: | Ensures the integrity of your samples |
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
AccuPower PCR Premix
Gentaur’s AccuPower PCR PreMix is a convenient lyophilized PCR master mix containing Top DNA Polymerase, dNTPs, reaction buffer, tracking dye, and patented stabilizer. AccuPower PreMix PCR master mix includes our super-processive “three times faster than Taq” Top DNA Polymerase for the fastest nucleic acid amplification. While Top DNA Polymerase is ideal for applications where you would normally use Taq, if your needs include HotStart PCR, or High Fidelity PCR, please see our AccuPower HotStart PCR and AccuPower TLA product pages. AccuPower PreMix PCR master mix is available with or without tracking dye, depending on your application. If purchased with tracking dye, reactions can be loaded on agarose gels without adding loading buffer.
Features and Benefits
| Ease of use: | Just add template and primers and start your PCR, dNTPs, buffer and enzyme are provided |
| Stability: | Stable at room temperature for a month and for 2 years in a -20°C freezer |
| Gel loading: | Available with or without a tracking dye for ease of use |
Specifications
5' to 3' exonuclease activity: No
3' to 5' exonuclease activity: No
Terminal transferase activity: Yes
Fragment Size: Up to 10 kb
Application
Standard PCR
Figure 1. Processivity comparison with Lambda DNA template and different PCR reagents.
M: 100 bp Plus DNA Ladder (D-1035)
A: Accupower PCR PreMix
B: Taq mastermix from Company S
C: Taq mastermix from Company T
Lane M: 1 kb DNA Ladder (D-1040)
Lane 1: 1 kb PCR product
Lane 2: 2 kb PCR product
Lane 3: 3 kb PCR product
Lane 4: 4 kb PCR product
Figure 2. Comparison of sensitivity test for AccuPower PreMix PCR master mix and other companies' products using serial diluted human genomic DNA.
Reaction condition: 95°C for 5 minutes, followed by 35 cycles of 20 seconds at 95°C, 20 seconds at 60°C, 30 seconds at 72°C.
A: human Insulin receptor gene
B: DNA cross-link repair 1A gene
I: AccuPower PCR PreMix
II: Taq DNA Polymerase from Company A
III: Taq DNA Polymerase from Company Q
IV: PCR PreMix from Company I
M: 100 bp DNA ladder (D-1030)
Lane 1: 10 ng
Lane 2: 1 ng
Lane 3: 100 ng
Lane 4: 10 ng
