Nov 24, 2016
SensiFAST™ Probe Hi-ROX Kit
- Bioline
- Bioline
External link: http://www.bioline.com/us/downloads/dl/file/id/2787/sensifast_probe_hi_rox_kit_manual.pdf
Protocol Citation: Bioline 2016. SensiFAST™ Probe Hi-ROX Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.fysbpwe
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 03, 2016
Last Modified: February 07, 2018
Protocol Integer ID: 3826
Abstract
The SensiFAST™ Probe Hi-ROX Kit has been developed for fast, highly reproducible real-time PCR and has been validated on commonly used real-time PCR instruments. The kit has been formulated for use with probe-detection technology, including TaqMan®, Scorpions® and molecular beacon probes. A combination of the latest advances in buffer chemistry and PCR enhancers, together with a hot-start DNA polymerase, ensures that the SensiFAST Probe Kit delivers fast, highly-specific and ultra-sensitive real-time PCR.
SensiFAST Probe is provided as a 2x mastermix containing all the components necessary for real-time PCR, including dNTPs, stabilizers and enhancers.
Guidelines
Kit Components
| Reagent | 200 x 20 μL reactions | 500 x 20 μL reactions | 2000 x 20 μL reactions | |
| SensiFAST Probe Hi-ROX mix (2x) | 2 x 1 mL | 5 x 1 mL | 4 x 5 mL |
Instrument compatibility
SensiFAST Probe Hi-ROX Kit has been optimized for use with all probe chemistries, including TaqMan, FRET, Scorpions and molecular beacon probes on real-time PCR instruments listed in the following compatibility table, each of these instruments having the capacity to analyze the real-time PCR data with the passive reference signal either on or off. The kit is also compatible with several instruments that do not require the use of ROX, such as the Mic (BMS), Qiagen (Corbett) Rotor-Gene™ 6000, the Bio-Rad CFX96 or the Roche LightCycler® 480.
| Manufacturer | Model | |
| ABI (Invitrogen) | 7000, 7300, 7700, 7900, 7900HT, StepOne™ and StepOne™ Plus |
General considerations
To help prevent any carry-over DNA contamination, we recommend that separate areas are maintained for reaction set-up, PCR amplification and any post-PCR gel analysis. It is essential that any tubes containing amplified PCR product are not opened in the PCR set-up area.
Primers and probe: These guidelines refer to the design and set-up of TaqMan probe-based PCR. Please refer to the relevant literature when using other probe types. The specific amplification, yield and overall efficiency of any real-time PCR can be critically affected by the sequence and concentration of the probes and primers, as well as by the amplicon length.
We strongly recommend taking the following points into consideration when designing and running your real-time PCR:
use primer-design software, such as Primer3 (http:// frodo.wi.mit.edu/primer3/) or visual OMP™ (http:// dnasoftware.com/). 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
optimal amplicon length should be 80-200 bp, and should not exceed 300 bp
final primer concentration of 400 nM is suitable for most Probe-based reactions, however to determine the optimal concentration we recommend titrating in the range 0.2-1 μM. The forward and reverse primers concentration should be equimolar
a final probe concentration of 100 nM is suitable for most applications; we recommend that the final probe concentration is at least 2-fold lower than the primer concentration
Note: In multiplex real-time PCR probe concentrations over 100nM can result in cross-channel fluorescence
Template: It is important that the DNA template is suitable for use in PCR in terms of purity and concentration. In addition, the template must be devoid of any contaminating PCR inhibitors (e.g. EDTA). The recommended amount of template for PCR is dependent upon the type of DNA used. The following points should be considered when using genomic DNA and cDNA templates:
Genomic DNA: use up to 1 μg of complex (e.g. eukaryotic) genomic DNA in a single PCR; we recommend using the Bioline ISOLATE II Genomic DNA Kit (BIO-52066) for high yield and purity from both prokaryotic and eukaryotic sources.
cDNA: the optimal amount of cDNA to use in a single PCR is dependent upon the copy number of the target gene; we suggest using 100 ng cDNA per reaction, however it may be necessary to vary this amount; to perform a two-step RT-PCR, we recommend using the SensiFAST cDNA Synthesis Kit (BIO-65042) for reverse transcription of the purified RNA; for high yield and purity of RNA, use the Bioline ISOLATE II RNA Mini Kit (BIO-52072).
MgCl2: The SensiFAST Probe mix contains an optimized concentration of MgCl2, it is not necessary to supplement the mix further.
PCR controls: It is important to detect the presence of contaminating DNA that may affect the reliability of the data. Always include a no-template control (NTC) reaction, replacing the template with PCR-grade water. When performing a two-step RT-PCR, set up a no RT control as well as an NTC for the PCR.
Optional ROX: The SensiFAST Probe Hi-ROX Kit is premixed with ROX (5-carboxy-X-rhodamine, succinymidyl ester), so that where necessary, ROX fluorescence can be optionally detected on certain real-time instruments. If your real-time instrument has the capability of using ROX and you wish to use this option, then this option must be selected by the user in the software.
Troubleshooting Guide:
See the Bioline full documentation for detailed troubleshooting instructions.
http://www.bioline.com/us/downloads/dl/file/id/2787/sensifast_probe_hi_rox_kit_manual.pdf
Materials
MATERIALS
SensiFAST™ Probe No-ROX KitBiolineCatalog #BIO-86005
Reaction mix composition
Reaction mix composition
Prepare a PCR mastermix. The volumes given below are based on a standard 20 μL final reaction mix and can be scaled accordingly.
| Reagent | Volume | Final Concentration | |
| 2x SensiFAST Probe Hi-ROX Mix | 10 μL | 1x | |
| 10μM Forward Primer | 0.8 μL | 400 nM | |
| 10μM Reverse Primer | 0.8 μL | 400 nM | |
| 10μM Probe | 0.2 μL | 100 nM | |
| Template | up to 8.2 μL | ||
| H2O | As required |
Sensitivity testing and Ct values
Sensitivity testing and Ct values
When comparing SensiFAST with a mix from another supplier we strongly recommend amplifying from a 10-fold template dilution series. Loss of detection at low template concentration is the only direct measurement of sensitivity. An early Ct value is not an indication of good sensitivity, but rather an indication of speed.
Suggested thermal cycling conditions
Suggested thermal cycling conditions
The real-time PCR conditions, in the table below, are suitable for the SensiFAST Probe Hi-ROX Kit with the amplicons of up to 200 bp. These cycling parameters have been optimized on a number of platforms, however they can be varied to suit different machine-specific protocols.
| Cycles | Temp. | Time | Notes | |
| 1 | 95°C | *2-5 min | Polymerase activation | |
| 40 | 95°C 60°C | 10s **20-50s | Denaturation Annealing/extension (acquire at end of step) |
*2min for cDNA, up to 5min for genomic DNA
**Up to 50s may be necessary for multiplexing with more than two probes