SYBR and Fluorescein Kit

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Catalogue No: abx461064
Price: US$826.50
(Size: 500 rxns)
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SYBR and Fluorescein Kit

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Datasheet SDS
Abbexa's SYBR and Fluorescein Kit consists of a ready-to-use 2X reaction mix containing all components required for fast, specific and sensitive RT-qPCR, including dNTPs, MgCl2, stabilizers, enhancers and an antibody-mediated hot-start DNA polymerase. The 500 rxns size is provided as 5 × 1 ml 2X SYBR and Fluorescein and the 2000 rxns size is provided as 4 × 5 ml 2X SYBR and Fluorescein Mix.

Target SYBR and Fluorescein 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 SYBR and Fluorescein Mix 10 µl
Forward Primer (10 µM) 0.8 µl
Reverse Primer (10 µM) 0.8 µl
Template Variable, up to 8.4 µl
Water Variable, up to 20 µl
Total Volume 20 µl

Thermal Cycling Conditions (2-Step):

Step Number of Cycles Temperature Time per Cycle
Polymerase Activation 1 cycle 95 °C 2 min (cDNA)
3 min (genomic DNA)
Denaturation40 cycles 95 °C 5 seconds
Annealing/Extension
(acquire at end of step)		60 °C 15-30 seconds
Not recommended to exceed 30 seconds

Thermal Cycling Conditions (3-Step):

Step Number of Cycles Temperature Time per Cycle
Polymerase Activation 1 cycle 95 °C 2 min (cDNA)
3 min (genomic DNA)
Denaturation40 cycles 95 °C 5 seconds
Annealing60-65 °C 10 seconds
Extension
(acquire at end of step)		72 °C 5-20 seconds
Not recommended to exceed 20 seconds

Notes:
  • Forward and reverse primers are generally used at a final concentration of 0.1-1 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. If required for optimization, it is recommmended to increase the concentration of primers in 100 nM increments. Primers should have a melting temperature (Tm) of approximately 60 °C.
  • 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 temperature below 60 °C. If the annealing temperature requires optimization, 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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