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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.

Are there specific recommendations for performing 1-step RT-qPCR on RNA isolated from clinical samples?

PCRAMP-021

The nucleic acid (either DNA or RNA) will most likely need to be purified from clinical samples due to sample/transport media specific inhibitors and RNAses. For example, for RNA purification, we recommend our sbeadex‚Ñ¢ Pathogen Nucleic Acid Purification Kit for optimal purification.

How do I calculate transformation efficiency (TE)?

TE = (# colonies) / (µg DNA transformed) * (final transformation volume) / (volume plated) * dilution factor = cfu / µg

For example, if you transformed 1 ng of plasmid DNA into cells, diluted the transformation to a final volume of 1000 µL with Recovery Medium, then plated 25 µL of a 1:100 dilution of that transformation, and the next day you counted 150 colonies on your plate, your calculation would look like this:

# of colonies: 150
µg DNA transformed: 0.001
Final tansformation volume = 1000 µL
Volume plated = 25 µL
Dilution factor = 1 µL into 100 µL, 100-fold (factor of 100)

TE = (150 / 0.001) * (1000 / 25) * 100 = 6 x 108 cfu / µg

There are transformation protocols for chemically competent cells that do not include a heat shock. Do I need to heat shock my competent cells?

We have not tested alternative transformation protocols extensively. You can avoid the heat shock step, but it is highly likely that your transformation efficiencies will decrease.

How do you determine the brightness of a dye?

The absolute intensity of a dye is a product of the extinction coefficient and the quantum yield. We have not measured the quantum yield for our dyes as this value is highly dependent upon the local environment, including the buffer system used for the measurement. However, we do provide the extinction coefficients for dye modifications at their lambda max wavelength, and these values are available under the Technical Specs tabs of our Oligo Modifications webpages. While quantum yield and extinction coefficients both contribute to dye detectability, the principal determinant for Stellaris® RNA FISH assays is actually the instrument optics, including the excitation source, available filters, and quantum efficiency of the camera.

Why are genotype calls not achieved for specific targets?

AMPSEQ-001
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 I use other dyes besides the Green Fluorescent Dye available in the LavaLAMP™ DNA Component Kit with Dye?

Yes. Syto-13 (Molecular Probes) and EvaGreen (Biotium) have both been tested with the LavaLAMP DNA Master Mix and they perform well.

Should I use electrocompetent or chemically competent cells to make my library?

We recommend using electrocompetent cells for library generation, because the transformation efficiency for electrocompetent cells is higher. Higher transformation efficiency preserves library diversity.

Which instruments can be used to monitor the real-time fluorescent assays?

See the User Manual for a list of instruments that have been tested at Biosearch Technologies. Basically, any instrument capable of maintaining stable reaction temperatures and detecting fluorescence in the FAM (fluorescein) channel will work.

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.