Probe Hi-ROX Kit

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Catalogue No: abx461058
Price: US$841.00
(Size: 500 rxns)
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Probe Hi-ROX Kit

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Datasheet SDS
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, MgCl2, 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)
Denaturation40 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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