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SleepyHollow02
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Untersuchte Arbeit:
Seite: 24, Zeilen: 1 ff. (komplett)
Quelle: Papa 2010
Seite(n): 29 f., Zeilen: 29: 4 ff.; 30: 1-7
Physical positions of the probes correspond to the UCSC genome browser - GRCh build 37, Feb 2009 (http://genome.ucsc.edu). DNA labelling was executed essentially according to the Agilent protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis 2.0v) using the Bioprime DNA labelling system (Invitrogen). Genomic DNA (2 μg) was mixed with 20 μl of 2.5X Random primer solution (Invitrogen) and MilliQ water to a total volume of 41 μl. The mix was denaturated at 95° C for 7 minutes and then incubated in ice/water for 5 minutes. Each sample was added with 5 μl of 10X dUTP nucleotide mix (1.2 mM dATP, dGTP, dCTP, 0.6 mM dTTP in 10 mM Tris pH 8 and 1 mM EDTA), 2.5 μl of Cy5-dUTP (test sample) or 2.5 μl of Cy3-dUTP (reference sample) and with 1.5 μl of Exo-Klenow (40 U/μl, Invitrogen). Labeled samples were subsequently purified using CyScribe GFX Purification kit (Amersham Biosciences) according to the manufacturer protocol. Test and reference DNA were pooled and mixed with 50 μg of Human Cot I DNA (Invitrogen), 50 μl of Blocking buffer (Agilent Technologies) and 250 μl of Hybridization buffer (Agilent Technologies). Before hybridization to the array the mix was denatured at 95°C for 7 minutes and then pre-associated at 37°C for 30 minutes. Probes were applied to the slide using an Agilent microarray hybridization station. Hybridization was carried out for 24/40 hrs at 65°C in a rotating oven (20 rpm). The array was disassembled and washed according to the manufacturer protocol with wash buffers supplied with the Agilent kit. The slides were dried and scanned using an Agilent G2565BA DNA microarray scanner. Image analysis was performed using the CGH Analytics software v.3.4.40 with default settings. The software automatically determines the fluorescence intensities of the spots for both fluorochromes performing background subtraction and data normalization, and compiles the data into a spreadsheet that links the fluorescent signal of every oligo on the array to the oligo name, its position on the array and its position in the genome. The linear order of the oligos is reconstituted in the ratio plots consistent with an ideogram. The ratio plot is arbitrarily assigned such that gains and losses in DNA copy number at a particular locus are observed as a deviation of the ratio plot from a modal value of 1.0. Physical positions of the probes correspond to the UCSC genome browser - NCBI build 36, March 2006. (http://genome.ucsc.edu). DNA labelling was executed essentially according to the Agilent protocol (Oligonucleotide Array-Based CGH for Genomic DNA Analysis 2.0v) using the Bioprime DNA labelling system (Invitrogen). Genomic DNA (2 μg) was mixed with 20 μl of 2.5X Random primer solution (Invitrogen) and MilliQ water to a total volume of 41 μl. The mix was denaturated at 95° C for 7 minutes and then incubated in ice/water for 5 minutes. Each sample was added with 5 μl of 10X dUTP nucleotide mix (1.2 mM dATP, dGTP, dCTP, 0.6 mM dTTP in 10 mM Tris pH 8 and 1 mM EDTA), 2.5 μl of Cy5-dUTP (test sample) or 2.5 μl of Cy3-dUTP (reference sample) and with 1.5 μl of Exo-Klenow (40 U/μl, Invitrogen). Labeled samples were subsequently purified using CyScribe GFX Purification kit (Amersham Biosciences) according to the manufacturer protocol. Test and reference DNA were pooled and mixed with 50 μg of Human Cot I DNA (Invitrogen), 50 μl of Blocking buffer (Agilent Technologies) and 250 μl of Hybridization buffer (Agilent Technologies). Before hybridization to the array the mix was denatured at 95°C for 7 minutes and then pre-associated at 37°C for 30 minutes. Probes were applied to the slide using an Agilent microarray hybridization station. Hybridization was carried out for 24/40 hrs at 65°C in a rotating oven (20 rpm). The array was disassembled and washed according to the manufacturer protocol with wash buffers supplied with the Agilent kit. The slides were dried and scanned using an Agilent G2565BA DNA microarray scanner. Image analysis was performed using the CGH Analytics software v. 3.4.40 with default settings. The software automatically determines the fluorescence intensities of the spots for both fluorochromes performing

[page 30:]

background subtraction and data normalization, and compiles the data into a spreadsheet that links the fluorescent signal of every oligo on the array to the oligo name, its position on the array and its position in the genome. The linear order of the oligos is reconstituted in the ratio plots consistent with an ideogram. The ratio plot is arbitrarily assigned such that gains and losses in DNA copy number at a particular locus are observed as a deviation of the ratio plot from a modal value of 1.0.

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