- One step RT-PCR
- RT-PCR of complex GC/AT rich templates
- Fast RT-PCR
- Includes both RT Mix and RT-PCR Mastermix for RT and PCR in one tube
- RT Mix contains RNase Inhibitor and thermostable RT (up to 55°C)
- RT PCR Mastermix allows for low copy number targets detection
highQu 1Step RT PCR Kit combines the 20X RT and RNase Inhibitor mix for efficient reverse transcription and the 2X PCR Mastermix for subsequent amplification of cDNA in the same tube.
RT2 Mix, 20X is a blend of the engineered MMuLV (stable at 40-55°C allowing for high yields of long transcripts) with an efficient Ribonuclease Inhibitor protecting the template RNA from RNases.
The resulting cDNA is then amplified by the 1Step RT PCR Mastermix, 2X. The PCR mastermix contains our proprietary Hot Start Taq DNA Polymerase. The enzyme activity at room temperature is blocked by small molecular inhibitor. Enzyme becomes active only after heating what allows for highly specific and extremely sensitive amplification, no primer dimer formation and no background. In combination with the optimized buffer the enzyme provides higher success rates in demanding PCR applications like amplification of complex or longer templates and fast cycling.
Hot Start Taq DNA Polymerase has the same PCR accuracy like Taq DNA Polymerase, 4.5 x 104 (nucleotides incorporated before the error occurs), and produces A-tailed products suitable for ligating into TA cloning vectors.
For the maximum convenience the Kit includes even the PCR Water to set up the reaction, so the only thing you need to take care of is the high quality RNA template.
- RNA is extremely sensitive to degradation by RNases present everywhere. Take care to protect RNA from degradation and to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
- Include a no-template control, no RT2 Mix control and positive control in parallel.
- Thaw and keep reagents on ice. Mix well before use.
- Perform cDNA synthesis 10 min at 45°C, 20 min for >1 kb, increase temperature to 55°C for complex templates.
- The longer the amplicon, the longer the extension: use 15 sec/kb for < 3 kb targets, increase time for >3 kb.
- Run an annealing temperature gradient from 58°C to 65°C to choose the best specificity conditions.
Prepare a 50 µl reaction:
|Rev. & For. Primers||variable, up to 0.4 µM final each (2 µl of 10 µM each)|
Total RNA or
|1 pg to 1 µg or
|PCR Water||to 22.5 µl|
|1Step RT PCR Mastermix, 2X||25 µl|
|RT2 Mix, 20X||2.5 µl|
- Mix gently, avoid bubbles.
- Place into the instrument set like:
|Reverse transcription||1 cycle: 45 -55°C – 10 to 20 min|
|Initial denaturation||1 cycle: 95°C – 2 min|
|Denaturation||40 cycles: 95°C - 10 sec|
|Annealing||40 cycles: 60-65°C – 10 sec|
|Extension||40 cycles: 72°C –30-60 sec (15 sec/kb)|
- After the PCR store probes shortly on ice, for long term store at -20°C.
of 50 µl
2 x 1.25 ml - 1Step RT PCR Mastermix, 2X
1X 1Step RT PCR Mastermix contains hot-start Taq DNA Polymerase, 0.25 mM dNTPs, 3 mM MgCl2, enhancers, stabilizers.
RT2 Mix is a 20X concentrated blend of reverse transcriptase and RNase inhibitor
In the dark at -20°C.