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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.

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.

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 BHQ Probe Master Mix be used for both end-point genotyping and qPCR applications?

BHQ-001

BHQ Probe Master Mix is a highly versatile PCR Master Mix, which has been validated for use with both endpoint genotyping and qPCR applications. Please refer to the BHQ Probe Master Mix User Guide for detailed instructions and protocols.

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.

Can BHQ Probe Master Mix be used with crudely-extracted DNA?

BHQ-001

Yes, BHQ Probe Master Mix has been optimised to with crudely-extracted DNA (Biosearch Technologies QuickExtract, HotShot) on a variety of agrigenomics samples (e.g. seeds, leaves), as well as on purified DNA. If working with agrigenomics samples, it is recommended to use dry material (e.g. desiccation of leaves using the Biosearch Technologies Plant Sampling Kit). This will allow for the most optimum recovery of DNA. Please contact our Technical Support team for further information.

What reaction volumes can be used with BHQ Probe Master Mix?

BHQ-001

BHQ Probe Master Mix has been validated for use in endpoint genotyping and qPCR applications in 1.6 µL – 25 µL reaction volumes.

What are the optimum thermal cycling conditions when working with BHQ Probe Master Mix?

BHQ-001

The thermal cycling conditions will depend various factors. For example, the annealing temperature will depend on the sequence of the target-specific primers and probes used in the reaction, and the number of cycles will be dependent on the efficiency of the PCR reaction. Please refer to the BHQ Probe Master Mix User Guide for further information on recommended protocols and adhering to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines.

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.

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