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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.

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.

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.

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.

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.

Are there any limitations on the types of fluorescent reporter molecules I can use with BHQ Probe Master Mix?

BHQ-001

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

Have you produced proteins at scale?

COMCEL-008

Our customers have reported growth of ClearColi strains in a 5 litre fermenter and shown optical density levels in excess of 150, and that some defoaming agents (particularly silicone-based defoamers) are beneficial while other defoaming agents such as polypropylene glycol may inhibit cell growth.

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.