Product FAQ

Do you have exon coverage in the high-resolution genes?

Answer

Yes, if using the Affymetrix™ defined workflow and depending on the size of the exon. (Our algorithm requires 20 contiguous markers to make a call.) If using an “open” workflow, the user can define the number of probes they need to claim exon resolution.

Answer Id: E13856

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Product FAQ

What algorithm(s) can be used to make copy number calls?

Answer

We recommend the TuScan algorithm. This algorithm has fixed settings based on 19 probes/call to ensure high sensitivity and specificity at the stated resolution claims. Nexus Express Software for OncoScan™ FFPE Assay Kit has an additional algorithm for copy number calls from OncoScan™ FFPE Assay Kit: SNPFASST2 algorithm, developed and supported by BioDiscovery. This allows the user to adjust settings and make custom calls (e.g., with fewer probes). Use cases are:
When breakpoints are clear with fewer probes
When probe re-centering is needed

Answer Id: E13857

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Product FAQ

What copy number calls do I get from TuScan?

Answer

Log2 and B-allele frequency.

Linear copy number calls when you mouse over aberrations. In some cases, linear calls are calls in aberrant cell fraction only. In the software, when the percentage of aberrant cells has a percentage value, the linear call for aberrations in that sample is the call in the aberrant cells only. Values will be integers. When the percentage of aberrant cells is “N/A,” the linear call is an average call across the aberrant and non-aberrant cells, and values will be non-integers. You can estimate the copy number in aberrant cells if you have an independent tumor burden estimate.

Answer Id: E13858

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Product FAQ

How are peptide/phosphopeptide antibody competition studies performed?

Answer

Invitrogen™ Phosphorylation Site-Specific Antibodies detect the phosphorylation of a particular amino acid residue within a particular protein. The immunogen used to raise a Phosphorylation Site Specific Antibody is a synthetic peptide, the sequence of which corresponds to the phosphorylated residue plus the surrounding residues of the protein of interest.  Because the immunogen corresponds to the phosphorylated amino acid residue within the context of the amino acid sequence of the protein, the antibody produced in response to the immunogen is specific to the phosphorylated protein, rather than to a phosphorylated amino acid residue.

Phosphorylation Site Specific Antibodies are useful for the detection of protein phosphorylation by Western blotting and other antibody based procedures, including immunohistochemistry, flow cytometry, and ELISA. The specificity of a Phosphorylation Site Specific Antibody for a phosphorylated protein can be verified by performing a simple peptide competition experiment. In this peptide competition experiment, the antibody of interest is pre-incubated with either (1) water (total signal control), (2) the phosphopeptide immunogen, or (3) the corresponding non-phosphopeptide.  The pre-incubated antibody is then used as the primary antibody in Western blotting or other antibody based procedure.  If the specificity of the antibody and the sample conditions are optimal, the phosphopeptide immunogen will be observed to block binding of the antibody to the phosphorylated protein, while the non-phosphopeptide will be observed to have no effect.

In most peptide competition experiments with Invitrogen™ control peptides, the phosphopeptide and non-phosphopeptide are used at a 200 fold molar excess over the antibody.  Please note that while a 200 fold molar excess of peptide to antibody is found to be satisfactory for most applications, use of a 500 fold molar excess of peptide to antibody may be optimal for a particular system. It is also important to note that with this and any antibody based procedure, some optimization of antibody concentration and incubation times may be necessary. Calculation for the determination of the 200 fold molar excess of peptide to antibody usually involves the following assumptions: The molecular mass of an IgG molecule is 150,000 daltons. Each mole of antibody binds two moles of peptide. The Phosphorylation Site Specific Antibody is used at a concentration of 0.5 mg/mL. The optimal antibody concentration for use in peptide competition experiments is a concentration which is below saturating as determined by previous experiments in your system.  If an optimal concentration has not been determined, it is suggested that the concentration provided on the antibody Product Analysis Sheet be used. 

Answer Id: E5122

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Product FAQ

What does the percentage of aberrant cells mean?

Answer

This represents the percentage of aberrant cells in a sample:

When reported as “N/A,” it means that the percentage of aberrant cells could not be estimated.
When reported as a percentage (e.g., 60%), it means that 60% of the cells were aberrant.

Answer Id: E13859

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Product FAQ

How can I export allelic difference, smooth signal, log ratio, and other probe-level data?

Answer

For a whole chromosome or a region, go to the Graphs tab in ChAS 3.1 and use the “copy to clipboard” or “export to txt” functions.

For the whole genome, using ChAS 2.1 or later versions, go to the analysis manager/analysis dashboard and select the “QC results” tab. Then open and/or select the file(s) to export, press the “Generate Report” button, and select the “Export probe level data function.” Data will be exported in .txt format.

Answer Id: E13875

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Product FAQ

Can I grow Gibco™ Human Astrocytes in standard tissue-culture vessels without matrix coating?

Answer

No. These cells must be grown in Geltrex™ Matrix-coated tissue culture vessels.

Answer Id: E12006

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Product FAQ

I did not recover any DNA. Why?

Answer

The DNA pellet from precipitation with isopropanol is easily dislodged when washing with 70% ethanol. It is best to remove the isopropanol supernatant and the ethanol wash by pipetting. Be careful not to shoot the washing buffer directly onto the pellet. Instead, allow the washing buffer to run over the pellet. Regardless of which manufacturer's miniprep kit you use, washing the pellet can be challenging because it is so small.

Answer Id: E5126

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Product FAQ

What is the efficiency of HDR (homology directed repair) after the double-stranded break?

Answer

HDR efficiency is very low, on average less than 2%.

Answer Id: E10125

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Product FAQ

Under what conditions can TuScan estimate the percentage of aberrant cell fraction?

Answer

This is possible when:

The tumor is nearly homogeneous (i.e., it has a major dominant clone), and the percentage of aberrant cell fraction is 40% or higher.

Answer Id: E13860

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Product FAQ

What are the recommended graph settings for data interpretation in ChAS 3.1?

Answer

We recommend changing the weighted log2 ratio graph from the default setting to minimum at -1.5 to maximum at 1.5, using data type as points. We recommend changing the allele peaks graph from the default setting to minimum at -2 to maximum at 2, using data type as points. The other graphs can stay at the default values.

Answer Id: E13876

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Product FAQ

Does the MagMAX™ mirVana™ Total RNA Isolation Kit come with plates?

Answer

Yes, it comes with one processing plate, two elution plates, and four plate covers to support manual processing. However, you will have to purchase deep-well plates, standard plates, and tip combs in addition to that if you wish to process on a KingFisher ™ instrument.

Answer Id: E10360

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Product FAQ

Where were Gibco™ Rat Primary Cortical Astrocytes (Cat. No. N7745) isolated from? How many passages do these cells go through before being frozen down?

Answer

Rat cortical astrocytes were isolated from the cortex of fetal (E-19) Sprague-Dawley rats. The cells were cryopreserved in growth medium supplemented with 10% DMSO at passage 1.

Answer Id: E12007

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Product FAQ

My plasmid yield was lower than expected. What could be the cause?

Answer

There are several reasons lower yields can occur:

-Make sure the binding of the plasmid is being done at RT. Temperature affects the pH of the binding solution.

-Make sure all other solutions are also warmed to RT for optimal yield.

-Verify that the centrifugation immediately following the neutralization step was not done at 4 degrees C. If it was, the supernatant MUST be warmed to RT before loading onto the column.

-The 1:1:1 ratio of buffers (Resuspension/Lysis/Precipitation) has not been kept precisely. A surplus of solution E3 will exceed the optimal salt concentration for DNA binding, thus leading to decreased DNA yield.

-Low-copy number plasmids give lower yields.

-If all of the medium was not removed at the cell harvesting step, the pH of the subsequent step can be affected and reduce yield.

-The cell pellet was not thoroughly resuspended before lysis.

-The purified DNA pellet was overdried after isopropanol precipitation and ethanol wash making it difficult to resuspend the pellet. Air dry only.

-Pellets can be easily lost during the alcohol precipitation and wash steps. It is best to pipette off the alcohol solutions rather than pouring them off, as the pellets tend to be slippery.

-After lysis and neutralization, the lysate is not homogeneous. After addition of solutions (Resuspension/Lysis/Precipitation buffers), cells must be mixed thoroughly by inverting the tubes until a homogeneous phase is obtained. Otherwise, cells will not be lysed completely and the plasmid DNA will stick to bacterial debris. In the case of bigger bacterial cell pellets, more vigorous shaking may be required to achieve homogeneity. However, do NOT vortex, as vortexing will release chromosomal DNA.

Answer Id: E5127

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Product FAQ

I am using the CRISPR technology and am worried about off-target effects. What is the best way to combat this issue?

Answer

Carefully designed crRNA target oligos and avoiding homology with other regions in the genome are critical for minimizing off-target effects.

Answer Id: E10126

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