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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 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.)

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.

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.

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.

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.