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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.

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 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.)

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 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).

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