1Step RT qPCR Probe kit - HighQu
highQu 1Step RT qPCR mastermixes in combination with a blend of thermostable and extremely active Reverse Transcriptase & advanced RNase Inhibitor (RT Mix) allow for a single step one tube RT qPCR.
Detailní popis
Applications
- RT-qPCR assays based on specific probes:
- TaqMan®, Molecular Beacons, Scorpions™ Probes
- Gene expression analysis, quantification of:
- gDNA, cDNA, viral DNA, low copy number genes
Benefits
- Efficient Reverse transcription & sensitive qPCR in one tube
- Both standard or fast cycling, GC rich templates
- Rapid extension, early Ct
- Supplied with PCR Water
highQu 1Step RT qPCR mastermixes in combination with a blend of thermostable and extremely active Reverse Transcriptase & advanced RNase Inhibitor (RT Mix) allow for a single step one tube RT qPCR.
qPCR mastermixes are based on the small molecular inhibitor technology Hot Start PCR allowing to achieve highest sensitivity and specificity under both standard and fast qPCR cycling conditions. They provide excellent results on both AT/GC rich templates and guaranty rapid extension with early Ct values with minimum or no optimization.
Our mastermixes are supplied with PCR Water to guaranty the best performance. To suit the broad instrument range the 1Step RT qPCR Probe Mixes are available in three versions – without ROX, with low or high ROX concentration.
Important Notes
- Take care to prevent RNA from degradation by widely spread and stable RNases.
- Prepare crude samples, set up reactions in different dedicated areas, use DEPC-treated nuclease-free labware and gloves.
- Before the cDNA synthesis check RNA quality on denaturing agarose gel to be sure you have good quality material.
- Use special primer selection programs for a good planning.
- Work with amplicons in a range of 80-200, max 400 bp
- Take typical measures to prevent PCR cross over contamination, keep your bench clean, wear gloves, use sterile tubes and filter pipet tips.
- Run reactions in triplets; include a no-template control, no RT Mix control and positive control in parallel.
- Thaw and keep reagents on ice. Mix well before use.
- Higher amounts of RT3 Mix improve Ct, but primer dimers may appear.
Prepare a 20 µl reaction:
| Reverse Primer | 100 - 400 nM final c. |
| Forward Primer | 100 - 400 nM final c. |
| Specific Probe | 200 nM final c. |
| Total RNA template or mRNA template |
1pg to 1 µg or >0.01 pg |
| PCR Water | to 10 μl |
| 1Step RT qPCR Mix, 2X | 10 μl |
| RT3 Mix, 20X | 1-2 μl |
- Mix gently, avoid bubbles.
- Place into the instrument set like:
| Reverse Transcription | 1 cycle: 40-55°C - 10 min |
| Initial denaturation | 1 cycle: 95°C - 2 min |
| Denaturation | 40 cycles: 95°C - 5 sec |
| Annealing/extension | 40 cycles: 60-65°C – 20-30 sec |
- Follow instrument instructions for melt curve analysis.
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