Probe Hi-ROX Kit
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Price:
US$841.00
(Size: 500 rxns)
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Abbexa's Probe Hi-ROX Kit consists of a ready-to-use 2X reaction mix containing all components required for fast, highly reproducible RT-qPCR, including dNTPs, MgCl
2, stabilizers, enhancers and a hot-start DNA polymerase. The 500 rxns size is provided as 5 × 1 ml 2X Probe Hi-ROX Mix and the 2000 rxns size is provided as 4 × 5 ml 2X Probe Hi-ROX Mix.
| Target |
Probe Hi-ROX Kit |
| Tested Applications |
PCR |
| Form |
Liquid |
| Storage |
Store at -20 °C. Avoid repeated freeze/thaw cycles. |
| Validity |
Up to 12 months. |
| Buffer |
The exact formulation is proprietary. |
| Availability |
Shipped within 3-7 working days. |
| Note |
This product is for research use only. Not for human consumption, cosmetic, therapeutic or diagnostic use. |
| Directions for use |
Reaction Components: | Component | Volume | | 2X Probe Hi-ROX Mix | 10 µl | | Forward Primer (10 µM) | 0.8 µl | | Reverse Primer (10 µM) | 0.8 µl | | Probe (10 µM) | 0.2 µl | | Template | Variable, up to 8.2 µl | | Water | Variable, up to 20 µl | | Total Volume | 20 µl | Thermal Cycling Conditions: | Step | Number of Cycles | Temperature | Time per Cycle | | Polymerase Activation | 1 cycle | 95 °C | 2 min (cDNA)
up to 5 min (genomic DNA) | | Denaturation | 40 cycles | 95 °C | 10 seconds | Annealing/Extension
(acquire at end of step) | 60 °C | 20-50 seconds | Notes: - Forward and reverse primers are generally used at a final concentration of 0.2-1 µM each. It is recommended to start with 400 nM as the final concentration. The final concentration of the forward primers should be the same as the final concentration of the reverse primers. Primers should have a melting temperature (Tm) of approximately 60 °C. The Tm of the probe should be approximately 10 °C higher than that of the primers. A final probe concentration of 100 nM is suitable for most applications; higher concentrations may result in cross-channel fluorescence in multiplex qPCR. It is recommended that the final probe concentration is at least 2-fold lower than the primer concentration.
- The amount of template in the reaction depends mainly on the type of DNA used. Up to 1 µg of complex (e.g. eukaryotic) genomic DNA can be used in a single PCR. For cDNA, it is recommended to use a starting amount of 100 ng cDNA per reaction, though this may need to be varied to optimize the assay. It is important that the template does not contain any PCR inhibitors such as EDTA.
- It is not recommended to use an annealing/extension temperature below 60 °C. If the annealing temperature requires optimization, increase or decrease it in steps of 2 °C.
- The extension time may need to be increased for amplification products over 200 bp, or reduced if non-specific products are observed.
- The optimal conditions will vary from reaction to reaction and are dependent on the system used. Each parameter needs to be adjusted by the end user and some optimization may be required.
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