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1. 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 analysis lots of different genes.
2. What is the advantage of working with a probe system?
The probe system is always specific; 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 optimisation than SYBR Green I assays.
3. Can I use a SensiMix SYBR Kit for a probe assay?
This is not possible because the buffer for SensiMix SYBR kits and the buffer for probe kits are different, and have been optimised for their specific assays.
4. Why is the reaction volume 25µl or 50µl for this product?
Most SensiMix reactions are 50µl with the exception of the SensiMix HRM Kit which is 25µl and SensiMix Capillary and SensiMix Capillary One-Step Kits which are 20µl. These are the volumes recommended by the machine manufacturers, and can be scaled up or down if desired (for example when using a 384 well plate).
5. Why do the SensiMix 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 10 minutes.
6. Why do you no longer sell kits contain dUTP (and UNG)?
This is because it is because it is only effective if all the researchers either doing PCR in the laboratory or using the same thermocycler are also 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.
7. Why do certain kits contain a ROX passive reference?
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.
8. Why are the ROX concentrations different?
The amount of the ROX passive reference dye needed varies depending on the instrument optics, our SensiMix kits have been optimised for these different instruments (see Product Selection Table).
9. 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 Table), the SensiMix SYBR & Fluorescein contains fluorescein premixed in the mastermix at optimised concentrations.
10. Is the SYBR in a separate tube?
For your convenience most of the SensiMix kits have an optimised amount of SYBR Green in the master mix. The exceptions are the SensiMix Capillary kits that have a separate vial of SYBR Green, so that it can be used for both probe-based and SYBR Green-based chemistries on the capillary instruments, the SensiMix Probe Kits that do not require SYBR and the SensiMix HRM which uses EvaGreen™ dye.
11. 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 pipeting 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 cDNA Synthesis Kit (BIO-65025) separately if you wish to take a two-step approach.
12. What template (RNA/cDNA) is this compatible with?
SensiMix 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 SensiMix 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)
For a more comprehensive list, please see our reference list.
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