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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).

Are there any limitations on the types of fluorescent reporter molecules I can use with BHQ Probe Master Mix?

BHQ-001

All fluorescent dyes listed on our Fluorophore and BHQ Selection Chart are available for use with BHQ Probe Master Mix, in which the corresponding BHQ dye has been identified.

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.

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.

What concentration of primers and probes should I used in my reactions?

BHQ-001

Please refer to the BHQ Probe Master Mix User Guide for detailed information and recommended protocols.

We recommend a final concentration of 900 nM of primer and 200 nM probe in each reaction for qPCR applications. Please note, that for multiplex reactions, further optimisation may be required.

If using BHQ Probe Master Mix for endpoint genotyping applications, we recommend final concentrations of 400 nM primer and 200 nM probe as starting concentrations during initial optimisation.

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