| Description |
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The OneStep RT-PCR Premix is a ready-to-use, 2X concentrated premix that contains all the reagents (except primers and template) needed for running RT-PCR in a single tube. Conventional RT-PCR kits employ a two step RT-PCR, which is performed sequential reactions and separately prepared reactions mixtures. These additional steps are undesirable, as they increase the risk of contamination.
One step RT-PCR premix minimizes the risk of contamination and increases the sensitivity of RNA amplification.
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| Content |
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2X One step RT-PCR Premix containing: |
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‧Hotstart Taq DNA polymerase.
‧Reverse transcriptases.
‧dNTP mix including dATP、dCTP、dGTP、dTTP
‧5mM MgCl2
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| OneStep RT-PCR Premix performance test |
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High sensitivity of RNA amplification by one step RT-PCR is confirmed by using 10 copies viral RNA as the template(amplified fragment: 0.5kb) |
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| Procedure |
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A. Preparation of the RT- PCR master Mix |
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- Preparing a master mix as follows:
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| Component |
Volume / reaction |
Final conc. |
| 2x One-step RT-PCR Premix |
12.5 μl |
1X |
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Forward Primers (10μM)
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variable |
0. 6~1.0 μM |
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Reversed Primers (10μM)
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variable |
0. 6~1.0 μM |
| H2O |
upto 23.0 μl |
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Mix the master mix thoroughly by pipetting up and down. |
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- dispense 23μl volumes into PCR tubes .
- Added 2μl of the RNA .
Mix carefully by pipetting up and down.
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B. Performing One Step RT- PCR |
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- Program your instrument as follows:
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| Reverse transcription : |
30 min |
50~60 ℃ |
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Initial PCR activation step :
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10 min |
95 ℃ |
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3-step cycling Denaturation :
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0.5 min |
94 ℃ |
| Annealing : |
0.5 min |
50~68 ℃ |
| Extension : |
1 min |
72 ℃ |
Number of cycle 35~40 cycles |
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- PCR tubes are still on ice. Wait until the thermal cycle has reached 50℃. Then place the PCR tubes in the thermal cycle and start the RT-PCR program
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