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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.

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.

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.