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What reaction volumes can be used with RapiDxFire qPCR 5X Master Mix GF?

PCRAMP-012

RapiDxFire qPCR Master Mix has been validated for use in qPCR applications in 1.6 µL – 25 µL reaction volumes.

Are there any limitations on the types of fluorescent reporter molecules I can use with RapiDxFire qPCR 5X Master Mix GF?

PCRAMP-012

All fluorescent dyes listed on our Fluorophore and BHQ Selection Chart are available for use with RapiDxFire qPCR 5X Master Mix GF, in which the corresponding BHQ dye has been identified.

Can RapiDxFire qPCR 5X Master Mix GF be used for both endpoint genotyping and qPCR applications?

PCRAMP-012

RapiDxFire qPCR 5X Master Mix GF has been validated to be highly sensitive in qPCR applications, for use in the clinical and molecular-diagnostic market. Its use for end-point genotyping has not been validated, however it may be used as such with the understanding that any data generated or inference of clinical results is subject to your own validation and quality control criteria thresholds.

What are the optimum thermal cycling conditions when working with RapiDxFire qPCR 5X Master Mix GF?

PCRAMP-012

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 RapiDxFire qPCR 5X Master Mix GF user guide for further information on recommended protocols and adhering to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines.

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).

Can RapiDxFire qPCR 5X Master Mix GF be used in multiplex reactions?

PCRAMP-012

Yes, RapiDxFire qPCR 5X Master Mix GF 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.)

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.

Is the NxSeq HT Dual Indexing Kit (Item. No. 15300-1) compatible with the NxSeq UltraLow DNA Library Prep Kit, 12 reactions (Item. No. 15012-2)?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

Yes. However if combining these two kits, you will be setting up fewer reactions, and you will need to add 2.5 µL aliquots of one 500 Series Primer and one 700 Series Primer to each library amplification reaction. Be extremely careful to avoid cross-contamination of the Indexed Primers when using only a portion of the supplied volume; open only one tube at a time, and ideally change gloves after closing one tube and before opening the next.

Are the NxSeq Single Indexing Kits (Item. No. 15100-1, 15200-1) compatible with the NxSeq UltraLow DNA Library Kit, 96 reactions (Item. No. 15096-2)?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

Yes. If you are doing lower multiplexing and only want/need single indexes, you can absolutely combine these kits. You will reduce the cost of your library prep by buying your library prep reagents in bulk.