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Which dyes are compatible with my thermal cycler?

BHQ-001

LGC, Biosearch Technologies offers many common fluorophores including FAM, HEX and TAMRA dyes, as well as our own proprietary dyes. Our CAL Fluor and Quasar dye series span the spectrum with emission wavelengths ranging from yellow to far-red, and represent alternatives to dyes such as VIC Cy3, Texas Red, LC Red ; 640, Cy5, and Cy5.5. For your convenience we have compiled Multiplexing Dye Recommendations Chart outlining optimal dye combinations in select qPCR machines, as well as a Fluorophore ; BHQ Dye Selection Chart listing reporter-quencher pairings. In addition, you may use our Spectral Overlay Tool to visualise the absorption and emission spectra of multiple dyes together.

Can intercalating dyes be used with this mix?

BHQ-001

BHQ Probe Master Mix has been optimised for use with hydrolysis-probe based chemistry, for both end-point genotyping and qPCR applications. For use with intercalating dyes, we would recommend that you perform your own optimisation and validation to ensure reproducible sensitivity and specificity is achieved using your selected dye.

Does the BHQ Probe Master Mix contain a passive reference dye (e.g. ROX)?

BHQ-001

Yes, BHQ Probe Master Mix contains the passive reference dye, ROX. BHQ Probe Master Mix is available with 3 differing levels of ROX (No ROX, Low ROX and Standard ROX), to allow for normalisation across a wide range of commercially available qPCR instruments.

If you are performing singleplex reactions, please see this table for the optimal ROX level for your qPCR instrument. For FRET-capable plate readers, Biosearch Technologies recommends initial trials with standard ROX BHQ Probe Master Mix.

If you are using the BHQ Probe Master Mix for multiplex reactions (using BHQ Dual-Labelled Probes, BHQplus, BHQnova and BHQplex CoPrimers), it is important to take your dye selection into account when deciding on the appropriate ROX level for the master mix. The ROX in the BHQ Probe Master Mix will occupy the same channel as CAL Fluor Red 610 and therefore, if this is one of the dyes used in your experiment, the master mix with no ROX will be the most suitable option. Our online can be used to check if your selected dyes overlap with ROX. Please see this table for our recommendations for multiplex experiments.

If our no ROX BHQ Probe Master Mix is the most appropriate for your experimental setup, but you do require normalisation, you could consider adding alternative normalisation dyes.

How do you determine the brightness of a dye?

The absolute intensity of a dye is a product of the extinction coefficient and the quantum yield. We have not measured the quantum yield for our dyes as this value is highly dependent upon the local environment, including the buffer system used for the measurement. However, we do provide the extinction coefficients for dye modifications at their lambda max wavelength, and these values are available under the Technical Specs tabs of our Oligo Modifications webpages. While quantum yield and extinction coefficients both contribute to dye detectability, the principal determinant for Stellaris® RNA FISH assays is actually the instrument optics, including the excitation source, available filters, and quantum efficiency of the camera.

How do I calibrate my instrument for the CAL Fluor® and Quasar® Dyes?

CAL Fluor® and Quasar® dye calibration standards are designed to improve the accuracy of signal detection in real-time thermal cyclers that require spectral calibration. They enable the instrument to store the fluorescence profile of each dye and control for channel cross-talk. Crosstalk is the bleed-through of fluorescent signal from a reporter into an adjacent filter or channel, an issue of particular concern in a multiplexed assay. Many qPCR machines are pre-calibrated for Cy™3 and Cy5 dyes. In those machines, no calibration is necessary to use our Quasar 570 (Cy3 alternative) and Quasar 670 (Cy5 alternative) dyes. To use our CAL Fluor dye labels, particularly in a multiplexing assay, certain real-time PCR instruments need to be calibrated to anticipate crosstalk. LGC Biosearch Technologies does not make available pure dyes. Instead, our calibration standards are formulated to better mimic a fluorescent probe under experimental conditions by covalently linking the dye to an oligo-thymidine (dT10).  A complete list of available Calibration and Reference Dyes is available through our website. Instructions to calibrate select qPCR machines are available in our Spectral Calibration Instructions.

Which dyes are compatible with my thermal cycler?

LGC Biosearch Technologies offers many common fluorophores including FAM, HEX and TAMRA dyes, as well as our own proprietary dyes. Our CAL Fluor® and Quasar® dye series span the spectrum with emission wavelengths ranging from yellow to far-red, and represent alternatives to dyes such as VIC®, Cy™3, Texas Red, LC Red® 640, Cy5, and Cy5.5. For your convenience we have compiled a Multiplexing Dye Recommendations Chart outlining optimal dye combinations in select qPCR machines, as well as a Fluorophore & BHQ® Dye Selection Chart listing reporter-quencher pairings. In addition, you may use our Spectral Overlay Tool to visualize the absorption and emission spectra of multiple dyes together.

Is BHQ Probe Master Mix stable at room temperature?

BHQ-001

BHQ Probe Master Mix is an incredibly stable PCR/qPCR Master Mix, which shows little-to-no reduction in performance when left at room temperature, even when combined with all other PCR-reaction components. We have demonstrated Cq > 1.0 when assembled qPCR reactions were left at room temperature for 16 hours, in both singleplex and multiplex applications.

To maintain high performance, it is recommended to store BHQ Probe Master Mix at -20 °C, and aliquot it into nuclease-free, light-protected tubes to minimise the number of freeze-thaw cycles. BHQ Probe Master Mix is also stable at +4 °C for 1 week.

What is the difference between lipid IVA and LPS?

BHQ-001

Lipid IVA is a modified LPS molecule that does not induce an endotoxic response in human cells. The six acyl chains of LPS (Figure 1) that trigger the endotoxic response are recognised by Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing NF-?B activation and production of proinflammatory cytokines.  Lipid IVA lacks the two secondary acyl chains and does not induce formation of the activated hTLR4/MD-2 complex, thus evading the endotoxic response.  In addition, the oligosaccharide chain is deleted, making it easier to remove the resulting homogenous lipid IVA from any downstream product.

ClearColi lipd IVA and LPS from unmodified E. coli

Figure 1:  Comparison of ClearColi lipid IVA and LPS from unmodified E. coli.  In ClearColi, two of the six acyl chains have been removed to disable the endotoxin signal, and the oligosaccharide chain has been deleted.

Why did you add dyes to the 500 Series and 700 Series Primers in the NxSeq HT Dual Indexing Kit?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

The goal was to help you ensure that each primer is correctly dispensed into your PCR amplification plate. By making each set of primers a different colour, you can verify that each one was added to every well of an entire row or column during PCR plate setup. We first recommend aliquoting 2.5 µL of each 700 Series Primer into each row of the plate using a 12-channel pipette. Each row should turn blue indicating that the primers were successfully dispensed. Second, we recommend aliquoting 2.5 µL of each 500 Series Primer (orange/yellow colour) into each column of the plate using an 8-channel pipette. As the 500 Series Primers are dispensed, each column on the plate should turn green indicating the successful addition of the 500 Series Primers (blue + orange/yellow = green).

Won’t the dyes in the primers affect my Illumina sequencing run and/or data quality?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

No. We’ve done extensive testing to demonstrate that the dyes do not affect the sequencing run/instruments or decrease the quality of the data generated. During the final library clean-up and size selection steps, the dyes are removed and therefore, do not make it on to the sequencer. To ensure compatibility in case of incomplete dye removal, we added the full concentration of dye found in the PCR amplification reactions to our final libraries and sequenced those samples on multiple instruments. Even with this artificially high concentration of dye in the denatured library (worst case scenario), we found no effect on the sequencing run/instrument performance or the yield and quality of the final data.