PCR master mix for high yield, homogeneous cDNA synthesis and amplification
AccuPower® CycleScript RT (available with: dT
20, dN
12, or dN
6 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 (dT
20, dN
12, or dN
6) PreMix series is stable for 3 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). Bioneer’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: Easy to use, simply add your purifies RNA
-
Broad range of working temperatures: For RNAs that are G:C rich or significant secondary structure
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Novel Cyclic Reverse Transcription: Ensures that you isolate even the rarest transcripts
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High yield of cDNA: For genes up to 9 kb within 10 minutes
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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)
Bioneer: Bioneer / 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 AccuPower® CycleScript 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