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How does the accuracy and sensitivity of the Qubit quantitation assays using the Qubit fluorometer compare to a microplate reader? Product FAQ

Answer

The accuracy and sensitivity of the Qubit quantitation assays are the same as that of a microplate reader. This was a requirement during product development. The detection limits for each Qubit kit can be found on the corresponding product manual, which can be found by searching our website by keyword or catalog number.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.

Answer Id: E5020

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What is the difference between the Quant-iT PicoGreen DNA, Quant-iT DNA, and Qubit DNA assays? Product FAQ

Answer

The Qubit Fluorometer contains highly optimized algorithms that calculate the concentration of the sample using either the Qubit assays or the Quant-iT DNA assays. The Quant-iT PicoGreen DNA assay may be adapted to the Qubit Fluorometer using the MyQubit firmware. The performance of all of these assays is similar.

The Quant-iT PicoGreen DNA assay is the most established assay and the most general-purpose (http://tools.thermofisher.com/content/sfs/manuals/PicoGreen-dsDNA-protocol.pdf). It requires the dilution of the standard DNA and buffer but can be adapted for use with either cuvettes, microplates, or the NanoDrop 3300.

The Quant-iT DNA assays provide a ready-to-use buffer and pre-diluted standard DNA for analyzing a large number of samples (>20 samples) using a 96-well microplate with no further adaptation.

The Qubit assays (https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-assays/myqubit.html) are intended for low throughput (<20 samples), and are only used on the Qubit Fluorometer.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Quantification Support Center.

Answer Id: E8129

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With the Oncomine Lung Cell-Free Total Nucleic Acid (cfNA) Research Assay, which method do you recommend for quantitating cfNA? Product FAQ

Answer

We recommend using the fluorescence-based Qubit dsDNA HS Assay Kit (Cat. No. Q32851) for quantification of cfNA and cfDNA samples. Spectrophotometric quantification methods are not recommended, because they are not reliable when the nucleic acid concentration is low. Use of these methods can lead to gross overestimation of the concentration of sample, under seeding of the target amplification reaction, and low library yields. There isn't a good absolute quantitation method for cfRNA.

Answer Id: E17081

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What is a shift assay? What is a supershift assay? Product FAQ

Answer

A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.

A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

Answer Id: E8002

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What is chromatin immunoprecipitation (ChIP)? Product FAQ

Answer

Chromatin immunoprecipitation (ChIP) assays identify links between the genome and the proteome by monitoring transcription regulation through histone modification (epigenetics) or transcription factor:DNA binding interactions. The strength of ChIP assays is their ability to capture a snapshot of specific protein: DNA interactions occurring in a system and to quantitate the interactions using quantitative polymerase chain reaction (qPCR). Chromatin IP experiments require a variety of proteomics and molecular biology methods including crosslinking, cell lysis (protein-DNA extraction), nucleic acid shearing, antibody-based immunoprecipitation, DNA sample clean-up and PCR. Additional techniques such as gel electrophoresis are usually used during optimization experiments to validate specific steps. See here for more details.

Answer Id: E15500

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Why is my library concentration low with the Ion AmpliSeq Microbiome Health Research Kit? Product FAQ

Answer

Here are possible causes and solutions:
-The sample DNA may have been misquantified. We recommend requantifying the sample DNA using the Qubit dsDNA HS Assay Kit and Qubit Fluorometer.
-The sample quality may have been low. We recommend reextracting the sample DNA using the MagMAX Microbiome Ultra Nucleic Acid Isolation Kit or adding more sample DNA to the amplification reaction.
-The PCR, digestion, or ligation reactions may have been inefficient. We recommend ensuring proper dispensing and mixing of viscous components at each step.
-The AMPure XP beads may have been overdried. We recommend not drying the AMPure XP beads for more than 5 mins.
Note: Library yield is not indicative of quality.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Answer Id: E18936

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I labeled my protein with a fluorescent dye. Do I really need to do the ‘degree of labeling' (DOL) determination? Product FAQ

Answer

We highly recommend that you determine the DOL. DOL determination is achieved by absorbance readings at either 260 nm (for nucleic acids) or 280 nm (for proteins) plus the absorbance maximum for the fluorophore. It is only by an absorbance reading that you may determine if the labeling reaction was successful and, if subsequent labeling of the same molecule is to be done in the future, to establish the best level of labeling for that molecule.
Fluorescence detection cannot tell you if the molecule is unlabeled, under-labeled, or overlabeled- all of these labeling scenarios may provide the same level of little or no fluorescence. With some dyes, overlabeling may result in dye-dye quenching, resulting in no detectable fluorescence, but the dye will be detected using absorbance. Also, visual inspection of the color of the solution of the labeled molecule cannot determine if it was suitably labeled.
A DOL measurement may be performed for biotinylated-molecules using either the HABA assay or similar assays (see Cat. No. B30751 FluoReporter Biotin Quantitation Assay Kit).

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Answer Id: E12210

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How should I measure cell-free RNA (cfRNA) yield from the MagMAX Cell-Free Total Nucleic Acid Isolation Kit? Product FAQ

Answer

cfRNA concentrations in plasma are low, and the conformation/length is not compatible with the dye in the Qubit RNA HS assay. We recommend using an m1 TaqMan assay with an appropriate amplicon length to detect cfRNA, such as Hs_99999905_m1 GAPDH (122 bases), Hs99999903_m1 ACTB 171 bases or another m1 assay target that is appropriate for your sample. The m1 designation indicates that the probe spans an exon-exon boundary ensuring that signal is only generated from a template with correctly spliced exons. The assay will not detect signal from the cfDNA that is also present in the eluate. We recommend using SuperScript VILO Master Mix (Cat. No. 11755050) for the RT step and the reaction volume for this step can be scaled down to 10 µL total, using 2 µL of purified cfNA input. We recommend performing the qPCR reaction according to the protocol for TaqMan Universal Master Mix II, no UNG (Cat. No. 4440040), with sample input up to 10% of the total reaction volume.

Answer Id: E16341

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Why can't I use a spectrophotometer to quantitate my cfDNA samples for the Oncomine Breast cfDNA Assay? Product FAQ

Answer

We do not recommend using spectrophotometer methods for quantifying cfDNA because they are not reliable with low concentrations of nucleic acids. Use of these methods can result in overestimation of the concentration of sample, under-seeding of the target amplification reaction, and low library yields.

Answer Id: E16361

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Why can't I use a spectrophotometer to quantitate my cfDNA samples for the Oncomine Breast cfDNA Research Assay v2? Product FAQ

Answer

We do not recommend using spectrophotometer methods for quantifying cfDNA because they are not reliable with low concentrations of nucleic acids. Use of these methods can result in overestimation of the concentration of sample, under-seeding of the target amplification reaction, and low library yields.

Answer Id: E16362

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My Xeno control assay failed. What are some possible causes? Product FAQ

Answer

If your Xeno control fails, you could possibly have inhibitors in your samples. You can test this by running the Xeno control directly in a qPCR reaction. You can also run a mock extraction control where you spike the Xeno control into a buffer sample and then perform the nucleic acid extraction procedure you used for your samples.

Answer Id: E16422

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Luminoskan Ascent Product Literature

Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2 Molecular Probes Handbook

Setting up a Photometric DNA Assay with SkanIt Software Using Pathlength Correction Product Literature

Microplate Based Pathlength Correction Method for Photometric DNA Quantification Assay Product Literature

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