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Locally averaged background correction

Manufactured by Bio-Rad

Locally-averaged background correction is a feature in Bio-Rad's lab equipment that adjusts the background signal in an image or data set. It calculates the local average of the background pixels and subtracts this value from the image or data to remove unwanted background interference.

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3 protocols using locally averaged background correction

1

Quantitative Analysis of ACB-PCR Products

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Following ACB-PCR, 10 Nl of bromophenol blue/xylene cyanol-containing 6× ficoll loading dye was added to each well of the 96-well plate, mixed, and 10 Nl of each ACB-PCR reaction product was loaded onto an 8% non-denaturing polyacrylamide gel. Fluorescein-labeled, ACB-PCR products of the correct-size were quantified using a PharosFX scanner with an external blue laser (Bio-Rad). Pixel intensities of the bands were quantified using Quantity One® software and a locally-averaged background correction (Bio-Rad).
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2

Quantifying Mutational Frequencies via ACB-PCR

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Equal volumes of ACB-PCR products were analyzed on non-denaturing 8% polyacrylamide gels, loading 10 μl of KRAS and BRAF ACB-PCR products or 15 μl of the PIK3CA and HRAS ACB-PCR products. The fluorescent ACB-PCR products were visualized using a PharosFX Molecular Imager with an external blue laser (Bio-Rad). Pixel intensities of the correct-sized bands were quantified using Quantity One software and a locally-averaged background correction (Bio-Rad). Although duplicate 10− 1 to 10− 5 MF standards were included in each experiment, the set of standards that gave the best quantification across the observable range of MFs for an entire set of unknown samples was used to construct each standard curve [10− 5 to 10− 1 for PIK3CA and BRAF, 10− 5 to 10− 3 for KRAS and HRAS]. For PIK3CA and KRAS, log-log plots relating MF to fluorescence (in pixels) were constructed and fit with a power function. For BRAF, log-linear plots relating MF to fluorescence were constructed and fit with a logarithmic function. For HRAS, linear-linear plots relating MF to fluorescence were constructed and fit with a linear function. Using the function of the standard curve and the pixel intensities of the ACB-PCR products for each mutation, the MF of each mutation within each unknown sample was calculated.
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3

Quantitative Analysis of ACB-PCR Products

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Equal volumes of ACB-PCR products were analyzed on nondenaturing 8% polyacrylamide gels. The fluorescent bands were visualized using a PharosFX Molecular Imager with an external blue laser (Bio-Rad). Pixel intensities of the bands (GAT ACB-PCR, 103 bp; GTT ACB-PCR, 89 bp) were quantified using Quantity One software and a locally averaged background correction (Bio-Rad). Log-linear plots relating MF to fluorescence (in pixels) were constructed and fit with a logarithmic function. For the GAT MF measurements, the average coefficient of determination (r2) for the standard curves was 0.9936 (n = 9, range 0.9876–0.9965). For the GTT MF measurements, the average coefficient of determination for the standard curves was 0.9945 (n = 9, range 0.9905–0.9973). This function was then used to calculate the MF in each unknown sample based on the fluorescence of its ACB-PCR product [45 ].
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