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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.

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.

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.

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, NAP40-032-00

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.

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.

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.

Which dyes are compatible with my thermal cycler?

LGC Biosearch Technologies offers many common fluorophores including FAM, HEX and TAMRA dyes, as well as our own proprietary dyes. Our CAL Fluor® and Quasar® dye series span the spectrum with emission wavelengths ranging from yellow to far-red, and represent alternatives to dyes such as VIC®, Cy™3, Texas Red, LC Red® 640, Cy5, and Cy5.5. For your convenience we have compiled a Multiplexing Dye Recommendations Chart outlining optimal dye combinations in select qPCR machines, as well as a Fluorophore & BHQ® Dye Selection Chart listing reporter-quencher pairings. In addition, you may use our Spectral Overlay Tool to visualize the absorption and emission spectra of multiple dyes together.

Does LGC Biosearch Technologies make Safety Data Sheets (SDS) available?

Safety Data Sheets (SDS), formerly referred to as Material Safety Data Sheets (MSDS), are available for download under the Technical Specs or Related Info tabs found on most product webpages. If you are unable to access this document or need additional information, please contact our Technical Support team.

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