SensiFAST™ SYBR® Lo-ROX Kit

Description

Outstanding assay reproducibility, sensitivity and robustness from both DNA and RNA templates, under fast thermal cycling conditions.

Product Highlights

Reproducible – consistent results between technical replicates for increased confidence in results
Specific – antibody-mediated hot-start DNA polymerase minimizes non-specific amplification for improved assay sensitivity and reliability
Sensitive – reliable quantification of low abundance targets and scarce samples
Robust – reliable, accurate detection of DNA and RNA targets from a broad range of sample types
Fast – delivers reproducible, accurate assay results in as little as 30 minutes

Product Description

The SensiFAST™ SYBR Lo-ROX Kit has been developed for fast, highly accurate real-time PCR and has been validated on all commonly-used real-time instruments that require a low concentration of the passive reference dye ROX.

A combination of the latest advances in buffer chemistry and PCR enhancers ensures that the SensiFAST SYBR Lo-ROX Kit produces reliable assay results under fast thermal cycling conditions. An antibody-mediated hot-start DNA polymerase system promotes highly-specific amplification, in turn improving assay sensitivity and dynamic range.

The SensiFAST SYBR® Lo-ROX Kit has been optimized to deliver optimal performance in tandem with the SensiFAST cDNA Synthesis Kit, which offers fast, unbiased cDNA synthesis, without compromising cDNA yield or coverage.

Highly efficient under fast conditions.

SensiFAST Products Customer Review

SensiFAST SYBR No-ROX One-Step Kit Customer Review

Real-Time PCR Selection Chart

One-step Vs. Two-step real-time RT PCR

A discussion of the pros and cons of each detection strategy.

I had a very good experience with the SensiFAST SYBR. It was quick and easy to use, while also providing me with near perfect R-squared values and dissociation curves. Even when compared to other, more expensive, master mixes, I obtained results of equal or better quality.

Mariah Meyer, University of Montana, DBS, US

Instrument Compatibility

Please refer to the Real-Time PCR Selection Chart to confirm the SensiFAST SYBR® Lo-ROX Kit is compatible with your instrument.

Resources

Documents

SensiFAST SYBR Lo-ROX Kit - MSDS SensiFAST SYBR Lo-ROX Kit - Product Manual

Real-Time PCR Selection Chart One-step Vs. Two-step real-time RT PCR

Certification of Analysis (COAs)

BIO-94050_SF884-B074810 BIO-94020_SF579-B074310 BIO-94005_SF578-B074020 BIO-94020_SF579-B070640 BIO-94050_SF884-B069560

ViewHide all 75 COAs

Specification

Components

Reagent	500 x 20µl Reactions	2000 x 20µl Reactions	5000 x 20 µL Reactions
SensiFAST SYBR^® Lo-ROX mix (2x)	5 x 1ml	4 x 5ml	10 x 5 mL

Volume

BIO-94005: 500 x 20µl Reactions: 5 x 1ml
BIO-94020: 2000 x 20µl Reactions: 4 x 5ml
BIO-94050: 5000 x 20 µL Reactions: 10 x 5 mL

Storage & Stability

All kit components should be stored at -20°C upon receipt for optimum stability. Repeated freeze/thaw cycles should be avoided. Avoid exposure of the SYBR^® Green I to light.

When stored under the recommended conditions and handled correctly, full activity of the kit is retained until the expiry date indicated on the outer box label.

Shipping conditions

Shipped on dry/blue ice.

FAQs

What is the difference between using ROX and using fluorescein?

Fluorescein is an alternative passive reference dye used just in the Bio-Rad instruments (see Product Selection Tool), the SensiFAST SYBR & Fluorescein contains fluorescein premixed in the mastermix at optimised concentrations.

Is the SYBR in a separate tube?

For your convenience the SensiFAST kits have an optimized amount of SYBR^® Green in the mastermix.

Can I use a SensiFAST SYBR Kit for a probe assay?

This is not possible because the SYBR^® is pre-mixed into the SensiFAST SYBR^® mastermix.

What template (RNA/cDNA) is this compatible with?

SensiFAST One-Step Kits can be used with most RNA/cDNA templates. To give you some idea of a few of the types of templates used with these kits:

Human - Rosato R.R., et al. Cancer Res. 67: 9490-500 (2007)
Rat - Remund K., et al. Expt. Lung Res. 35: 359-370 (2009)
Drosophila - Brown A.E., et al. PloS ONE 4: e4490 (2009)
Nitrogen-fixing bacteria - Bahlawane C., et al. Mol. Plant-Microbe Inter. 21: 1498-1509 (2008)
Diphtheria bacteria - Jochmann N., et al. Microbiology 155: 1459-1477 (2009)
Green algae - Wobbe L., et al. PNAS 106: 13290-13295 (2009)
Bovine virus - Park S-I., et al. J. Virol. Methods. 159: 64-6 (2009)

For cDNA/DNA templates SensiFAST SYBR^® and Probe kits are used. To give you some idea of a few of the types of templates used with these kits:

Stem Cell - Bernardo A.S., et al. Stem Cells 27(2): 341 -351 (2008)
Cow - Baumert A., et al. J. Dairy Research 76(3): 356-364 (2009)
Mouse - Hoyles R.K., et al. Arthritis Rheum. 58(4): 1175-88 (2008)
Quail - De Winter P., et al. British Poultry Science 49(5): 566 – 573 (2008)
Insect - Bass C., et al. Malaria journal 6 111 (2007)
Fish - Miller M.R., et al. J. Nutr. 138(11): 2179-2185 (2008)
Crab - Wilcockson D.C. & Webster S.G. Gen. & Comp. Endo. 156: 113–125 (2008)
Nematode - Murray S.L., et al. Mol. Plant-Microbe Interactions 20(11): 1431–1438 (2007)
Plant - Hecht V., et al. Plant Physiology Preview DOI:10.1104/pp.107.096818 (2007)
Yeast - Kawauchi J., et al. Genes & Dev. 22: 1082-1092 (2008)
E.coli Bacteria - Saeed H.A., et al. Research J. Microbiology 4(4); 173-177 (2009)
Virus - Muscillo M., et al. Water, Air, & Soil Pollution 191: 1-4 (2008)

Can I use a SensiFAST Kit for standard real-time PCR?

Although SensiFAST kits have been designed for fast PCR on the new generation of fast machines, they will work equally well for standard or fast PCR protocols on all real-time PCR machines.

Why do the SensiFAST kits contain a hot-start Taq polymerase?

Polymerase activity during the reaction set-up causes non-specific amplification including primer-dimer formation. To avoid non-specific amplification the polymerase is only activated after heating at 95°C for 2-3 minutes.

I would like to compare SensiFAST with my current qPCR mix. What do I have to consider?

When comparing SensiFAST with a mix from another supplier we strongly recommend to use the suggested conditions for PCR setup and cycling. Every real-time mix has its specific composition and different properties. That’s why they should be tested with their specific conditions, mostly this is possible on different plates only. We recommend amplifying from a ten-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.

Why do certain kits contain a ROX?

The emission recorded from ROX during the baseline cycles is used to normalize the emission recorded from the reporter (SYBR^®) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.

Why are the ROX concentrations different?

The amount of the ROX passive reference dye needed varies depending on the instrument optics. Our SensiFAST kits have been optimized for all these different instruments (see Product Selection Tool).

What could be done to avoid unspecific products in melting curve analysis?

Multiple products in melting curve analysis may be caused by unspecific products or primer dimer. However, appearance of multiple bands in melting curve analysis does not necessarily show different products. Multiple peaks may also appear, if distinct regions of the PCR product melt at different temperatures (e.g. due to an inconsistent GC content). We recommend to analyze the PCR product on a gel, to decide if unspecific products are present. The troubleshooting guide of your product insert includes several suggestions to solve these problems. In many cases problems with non-specific products can be solved if the annealing temperature is increased and/or the elongation time is decreased. If you have further questions, please contact Bioline Technical Support.

What is the advantage of working with SYBR Green I?

SYBR^® Green I is an inexpensive, universal dye which binds to all dsDNA. It can be easily used in combination with a simple primer pair to detect PCR products in real-time. This dye is very attractive for researchers analysing lots of different genes.

What is the advantage of working with a probe system?

The probe system is always specific, probes only detect the gene of interest. It is also possible to distinguish between similar sequences with small differences like SNPs or mutations. In general, probe assays need less optimization than SYBR^® Green I assays.

Why do you not sell kits contain dUTP (and UNG)?

This method is only effective if all the researchers in the laboratory, or using the thermocycler, are all using a dUTP/dNTP and UNG system. If even one researcher is not, it ceases to be an effective control. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency. Most labs do not need to use the dUTP method of control, optimised protocols will allow high specificity of PCR and good lab practices (using disposable consumables, the use of filter tips and maintaining a separate area for PCR set-up and PCR amplification and any post-PCR analysis) virtually eliminate the risk of cross contamination.

What is the difference between a one-step and a two-step real-time PCR reaction?

In a one-step reaction the reverse transcription reaction and the real-time PCR reaction are done in one tube, making this a closed tube assay, so contamination can be avoided. It saves pipetting steps and time and is easy in handling, making it ideal for high throughput screening. In a two-step reaction the reverse transcription reaction and the real-time PCR reaction are done in separate tubes. It gives a more flexible way of working in that the cDNA can be used for more than one real-time PCR reaction and can be archived, eliminating the need to continually isolate RNA. For convenience Bioline sells the SensiFAST cDNA Synthesis Kit separately if you wish to take a two-step approach.

What could be the reason of strange shaped qPCR curves? I observed an early increase of the signal and strange shaped qPCR curves without an initial plateau.

A possible reason is that the template amount is too concentrated. An early Ct may impair the baseline correction and may cause these strange curves. We suggest to dilute the template. Alternatively if the curve is flattened, this may be due to inhibitors in the reaction. Running a serial dilution of your sample will dilute the inhibitor concentration, if this corrects the problem, you will either need to go back and re-purify your template, or use it at a lower concentration, where the inhibitor effect has been diluted out.

Reviews

"We trialled the SensiFast SYBR LoRox on a QuantStudio real time PCR machine ... "

Raewyn Poulsen, University of Auckland, Faculty of Medicine, New Zealand

"I used SensiFAST SYBR Lo-ROX kit on my murine cDNA samples on an ABI 7500 ... "

So Ri Jung, School of Life and Environmental Sciences, The University of Sydney, Australia

"After multiple years using products from other companies, we have now ... "

Sean Wilson, Murdoch Childrens Research Institute, Parkville, Australia

"I had a very good experience with the SensiFAST SYBR Lo-ROX qPCR master mix. ... "

Mariah Meyer, The University of Montana, DBS, US

""We applied the SensiFAST SYBR Lo-ROX Kit to investigate SFRP4 ... "

Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, Sydney, Australia

"We applied the SensiFAST SYBR Lo-ROX Kit to investigate SFRP4 expression in primary and immortalized normal and cancer cell lines. We found that SensiFAST performed in a more sensitive way compared to two other real-time qPCR suppliers detecting SFRP4 expression one to two cycles earlier."
Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, Sydney, Australia
I had a very good experience with the SensiFAST SYBR Lo-ROX qPCR master mix. It was quick and easy to use, while also providing me with near perfect R-squared values and dissociation curves. Even when compared to other, more expensive, master mixes, I obtained results of equal or better quality.

Mariah Meyer, The University of Montana, DBS, US
After multiple years using products from other companies, we have now trialed and switched to using Bioline products for many aspects of our work. We use the Bioline RNA extraction kits, SensiFast cDNA synthesis and SYBR Lo-Rox kits for our transcriptional analysis and are very happy with the results this provides. The sensitivity is as good as if not better than other products, and is more cost beneficial as well. The rep we deal is always available to help us in any way we need, which helps makes our experience with Bioline simple and effective.
Sean Wilson, Murdoch Childrens Research Institute, Parkville, Australia
I used SensiFAST SYBR Lo-ROX kit on my murine cDNA samples on an ABI 7500 FAST instrument and I’m happy to say that it provides great sensitivity and reproducibility. Also, the reasonable price allows me to thoroughly optimise my reactions.
So Ri Jung, School of Life and Environmental Sciences, The University of Sydney, Australia
We trialled the SensiFast SYBR LoRox on a QuantStudio real time PCR machine using primers supplied by Qiagen. In a side-by-side comparison with two other SYBR green master mixes from different suppliers, we found SensiFast gave us lower cT values and lower variation between replicates. We also found we could detect the presence of a less abundant transcript in samples when we had used SensiFast, but not when we had used our two other SYBR master mixes.
Raewyn Poulsen, University of Auckland, Faculty of Medicine, New Zealand

Citations

1

SNP Microarray analyses reveal copy number alterations and progressive genome reorganization during tumor development in SVT/t driven mice breast cancer

Standfuß C., Pospisil H., Klein A. 2

Loss of Secreted Frizzled-Related Protein 4 Correlates with an Aggressive Phenotype and Predicts Poor Outcome in Ovarian Cancer Patients

Jacob F., Ukegjini K., Nixdorf S., Ford C.E., Olivier J., Caduff R., Scurry J.P., Guertler R., Hornung D., Mueller R., Fink D.A., Hacker N.F., Heinzelmann-Schwarz V.A. 3

Oxygen-dependent phenotypic variation in group A streptococcus

Chiang-Ni, C., Nian, S.Y., Wu, J.J., Chen, C.J. 4

Linking stomatal traits and expression of slow anion channel genes HvSLAH1 and HvSLAC1 with grain yield for increasing salinity tolerance in barley

Liu, X., Mak, M., Babla, M., Wang, F., Chen, G., Veljanoski, F., Wang, G., Shabala, S., Zhou, M., Chen, Z.H. 5

Mating-type switching by chromosomal inversion in methylotrophic yeasts suggests an origin for the three-locus Saccharomyces cerevisiae system

Hanson, S.J., Byrne, K.P., Wolfe, K.H.