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How do I calibrate my instrument for the CAL Fluor® and Quasar® Dyes?

CAL Fluor® and Quasar® dye calibration standards are designed to improve the accuracy of signal detection in real-time thermal cyclers that require spectral calibration. They enable the instrument to store the fluorescence profile of each dye and control for channel cross-talk. Crosstalk is the bleed-through of fluorescent signal from a reporter into an adjacent filter or channel, an issue of particular concern in a multiplexed assay. Many qPCR machines are pre-calibrated for Cy™3 and Cy5 dyes. In those machines, no calibration is necessary to use our Quasar 570 (Cy3 alternative) and Quasar 670 (Cy5 alternative) dyes. To use our CAL Fluor dye labels, particularly in a multiplexing assay, certain real-time PCR instruments need to be calibrated to anticipate crosstalk. LGC Biosearch Technologies does not make available pure dyes. Instead, our calibration standards are formulated to better mimic a fluorescent probe under experimental conditions by covalently linking the dye to an oligo-thymidine (dT10).  A complete list of available Calibration and Reference Dyes is available through our website. Instructions to calibrate select qPCR machines are available in our Spectral Calibration Instructions.

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 Amp-Seq provide data analysis software to generate genotype calls?

AMPSEQ-001
Biosearch Technologies provides an analysis pipeline (the BiosearchCaller software), compatible with the Amp-Seq technology, to produce genotypes from the sequencing data. The software can be accessed via a cloud system and our technical team will support the deployment and initial setup.

Can Amp-Seq One technology be used in human clinical diagnostics?

AMPSEQKIT1
Currently, we are unable to support human clinical diagnostic applications.

Does Amp-Seq One provide data analysis software to generate genotype calls? 

AMPSEQKIT1
Biosearch Technologies provides an analysis pipeline (the BiosearchCaller software), compatible with the Amp-Seq One technology, to produce genotypes from the sequencing data. The software can be accessed via a cloud system and our technical team will support the deployment and initial setup.

Why are genotype calls not achieved for specific targets?

AMPSEQKIT1

Sometimes, despite a successful primer design for a target, genotyping calls are not achieved for a specific target. This can be caused by several issues including poor primer design and issues with the synthesis of the primers. In addition, if genomic reference data for both parental strains is not provided for target primer design, some primer pairs might not work for all F1 samples.

Missing genotypes are generally attributed to low depth. Certain targets cannot be amplified because of sample quality issues or unknown parental bias in the genomes. Our standard design process addresses the parental bias by avoiding primers on known variability in the genome. Known variation information can be provided to us in the target flanking sequences through the Data Submission form.

Can Amp-Seq technology be used in human clinical diagnostics?

AMPSEQ-001
Currently, we are unable to support human clinical diagnostic applications.

Do you have any recommendations for purifying DNA from difficult biological samples such as plant leaves and seeds before shearing?

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

No. We do not have any specific recommendations for DNA purification. However, the purified DNA should be high quality with A260/280 ratios above 1.8. The DNA should also be free of detectable RNA contamination. Prior to shearing/fragmentation, the purified DNA samples can be analysed on 0.7% agarose gel to check for sample degradation and RNA contamination.

Are Endura High-Yield Electrocompetent Cells classified as genetically modified organisms (GMO)?

COMCEL-017
Depending on the definition of GMO, yes, they are GMO as they were constructed by methods outside of E. coli conjugations or viral transductions. However, they do not contain DNA from organisms other than E. coli.

What purification scale is sbeadex Lightning suitable for?

SBXLCK, SBXLCKSTART

The standard protocol assumes a 200 µL volume of lysate for each purification. The purification protocol can be upscaled or downscaled depending on laboratory requirements. We do not provide different scales of kits (e.g. mini, maxi); instead core kit reagents and additional reagents can be adjusted to suit your specific laboratory process.