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Wed, 10 Mar 2010 19:15:07 +0000
| Dyes |
How do I calibrate my instrument for the CAL Fluor Dyes?
In order to successfully use dual-labeled probes containing CAL Fluor® and/or Quasar® dyes for real-time PCR and quantitative PCR (qPCR) multiplex assays, certain real-time PCR instruments need to be calibrated to recognize the pure dye spectra. Spectral calibration is critical for multiplexed assays to differentiate overlapping fluorescent signals from one another. The document below with give users step-by-step instructions for calibrating their instruments:
/assets/BTI_Spectral_Calibration_Instructions.pdf
How do I know what fluorophore to pick for my dual-labeled probe?
The choice of fluorescent reporter to label your dual-labeled probe(s) depends upon your instrument optics and also the degree of multiplexing you wish to achieve.
If your assays will be amplified separately and not combined together into a multiplexed arrangement, it would be recommended to label each with FAM. FAM is the most commonly used fluorophore and is nicely detected by all real-time PCR instruments.
The optic capabilities of the instrument principally determines the degree of multiplexing and which fluorophores should be used. The following compatibility chart and multiplexing guide can be used to determine this:
http://www.biosearchtech.com/support/applications/multiplexing-qpcr.aspx
/download/brochures/bti_bhq_selectionchart.pdf
Why is BHQ-3 not an option when selecting Quasar or Pulsar dye labels?
Although we do offer BHQ-3 as a modification for oligos, we do not offer it paired to Pulsar 650, Quasar 670 or Quasar 705. For those dyes, we recommend BHQ-2. We do have data showing that BHQ-2 would be the optimal quencher to use. If you look on the Biosearch website under the Dual-labeledes, you will see that we automatically paired Pulsar 650, Quasar 670 or Quasar 705 with BHQ-2.
We generally do not recommend using BHQ-3 for Dual-labeledes since we cannot guarantee its results (minimum yield, functional performance, etc). It may be somewhat misleading when you look at the spectra of BHQ-3 compared to Quasar 670 because it is illustrating the traditional model of quenching and spectral overlap. It is possible that the principle quenching mechanism is not FRET but rather static quenching. A hydrophobic attraction between the fluorophore and quencher can promote the formation of an intramolecular dimer, which is non-fluorescent.
Scientists at Biosearch have characterized this quenching mechanism in several papers.
Does Biosearch have a list of all dyes available for DNA labeling?
Biosearch carries a variety of commonly used dyes as well as proprietary dyes.
Please refer to the following link for a complete listing of various dyes/fluorophores we carry at Biosearch:
/assets/bti_bhq_selectionchart.pdf
The dyes indicated in BOLD font are the dyes that we carry at Biosearch. If you have any questions regarding availability of dyes, please contact our Technical Support department at techsupport@biosearchtech.com
Does Biosearch Technologies offer NED fluorophores?
Biosearch Technologies does not offer NED™ dye because it is a proprietary dye owned by Applied Biosystems, Inc. (part of Life Technologies). We do, however, offer alternative dyes depending on your application. Many longer-wavelength dyes do not perform well in fragment analyzers such as the 3730 because these instruments rely upon a single 488 nmol laser for excitation and poorly excites red-shifted dyes. Applied Biosystem’s solution to this problem is to partner red dyes such as NED with FAM in a FRET construct. Biosearch does not offer such constructs so instead we advise customers to test our dyes on their instruments first. Should you decide to experiment with a dye that we manufacture, we recommend that you substitute NED with TAMRA.
Please refer to our Dye Selection Chart to review the dyes offered at Biosearch Technologies: /download/brochures/bti_bhq_selectionchart.pdf
Which dyes are compatible with my thermal cycler?
At Biosearch, we carry both commonly used dyes such as FAM and TAMRA as well as dyes we have developed the Quasar® and CAL Fluor® dyes, because the optics and filters of thermal cyclers vary. We have compiled a chart detailing dye compatibility which can be found here: http://www.biosearchtech.com/support/applications/multiplexing-qpcr.aspx
You can also download a PDF of our instrument-dye compatibility chart which also includes a dye selection chart.
What is the difference between Black Hole Quenchers (BHQ) and TAMRA?
There are significant advantages in using Black Hole Quenchers (BHQ) compared to other commonly used quenchers such as TAMRA. One of the main advantages is that BHQs are dark quenchers and will not emit fluorescence thereby decreasing background fluorescence and increasing the signal to noise ratios when compared to corresponding TAMRA probes. Using BHQs also allows more flexibility in terms of dye choices for multiplexing. The BHQ family readily permits single-tube multiplexing due to the increased variety of reporter dyes than can be effectively quenched with little or no cross-talk between reporters. The broader spectral coverage of the BHQ dyes provides the scientist with a larger pool of distinct, spectrally resolved reporter dyes which simplifies the design, implementation and interpretation of multiplexed hybridization probe assays. Visit our Black Hole Quencher dye webpage for more information.
What is the difference between Biosearch’s Quasar® dyes and the Cy™ dyes?
The Quasar dyes are direct replacements for the Cy™ dyes. The core structure of the Quasar dyes is identical to the core structure of the Cy™ dyes. Therefore, the Quasar dyes have the same spectral properties and perform equivalently to the Cy™ dyes and thus are direct replacements for their respective Cy™ dye equivalent (Quasar 570 for Cy3™ and Quasar 670 for Cy5™). The Quasar dyes do differ slightly in their solubility in the reagents and solvents utilized for DNA synthesis. They have been modified slightly with non-reactive groups that affect their solubility. Our modifications are sufficiently different such that they are not covered by patents from other companies. Also, the Quasar dyes are available as amidites, rather than succinimidyl esters, which eliminates the significant time and resource requirements of hand labeling required with the Cy™ dyes. For additional information, such as comparisons between the spectral properties of the Cy™ dyes and the Quasars, please email techsupport@biosearchtech.com
How do you determine the brightness of a dye?
The relative brightness of a dye is calculated by multiplying the extinction coefficient by the fluorescence quantum yield. However the relative brightness is also largely determined by the instrument used. Depending on the instruments’ filters, certain dyes may be easier to detect than others, causing different results to be seen depending on the configuration of the real-time PCR machine.
What are the quantum yields of the Black Hole Quenchers?
A quantum yield of fluorescence can be between 0 and 1 or 0% and 100%. The quantum yield of fluorescence is a ratio of how many photons are emitted per photon absorbed. A perfect fluorophore has a quantum yield of 1 or 100%. The BHQs are true dark quenchers and do not emit light. Therefore the quantum yield is close to zero.
Are Biosearch dyes compatible with DNA sequencing or fragment analyzers?
Biosearch dyes have not been tested for compatibility in these instruments. While the potential is there, dye substitution is made more complicated by the fact that many DNA sequencers are calibrated with FRET constructs composed of two dyes such as FAM/NED, though the construct is named only for one dye in the pair. In this example the calibrant would be named, NED.
Biosearch dyes are principally intended for use in probe hybridization studies such as qPCR and SNP genotyping. Using appropriate calibration methods, Biosearch dyes can be used with most real-time PCR machines.
