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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.

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.

What is the difference between RapiDxFire™ qPCR 5X Master Mix GF and BHQ™ Probe Master Mix?

PCRAMP-012

RapiDxFire qPCR 5X Master Mix GF is a 5X qPCR master mix produced and purified in a quality standard ISO 13485-certified manufacturing facility, allowing for its use in molecular diagnostic and clinical applications.

BHQ Probe Master Mix is a 2X PCR master mix, which has been validated for both endpoint genotyping and qPCR applications. It has been designed specifically to be inhibitor-tolerant, especially when working with crude-extracted agrigenomic samples (e.g. leaves, seeds).

Does the RapiDxFire qPCR 5X Master Mix GF contain a passive reference dye?

PCRAMP-012

No, the RapiDxFire qPCR 5X Master Mix GF does not contain a passive reference dye. However, customers can supplement their reaction mix with a passive reference dye of their choice.

What are the storage conditions for BHQ Probe Master Mix?

BHQ-001

BHQ Probe Master Mix should be stored at -20 °C. Repeated freeze-thaws should be avoided, so if BHQ Probe Master Mix is to be aliquoted, nuclease-free, light-protected tubes/vials should be used.

Once thawed, BHQ Probe Master Mix can be stored at +4 °C for 1 week.

Can BHQ Probe Master Mix be used in multiplex reactions?

BHQ-001

Yes, it has been optimised for use in multiplex qPCR applications and customers can chose from a wide range of fluorophores from Biosearch Technologies, depending on their qPCR instrument.

When performing multiplexing reactions, we recommend optimising each assay as a singleplex reaction, before combining a multiplex, to evaluate the optimal parameters of each assay (e.g. annealing temperature, cycling conditions etc.)

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