Biomarker Discovery:Article Title: High-throughput variant detection using a color-mixing strategy
Article Snippet: Dilution of gDNA samples and synthetic DNA templates were made in 1× TE buffer with 0.1% Tween 20 from Sigma Aldrich. .. For the tuberculosis color-mixing validation, with the assumption that clinically extracted genomic DNA of Mycobacterium tuberculosis from sputum specimen is low in amount and also contains some human cells or human genomic DNA, the WT sample was a mixture of 2 ng/μL of human cell-line gDNA 18572 (Coriell Biorepository) and 1,000 copies/μL of H37Rv wildtype Mycobacterium tuberculosis genomic DNA (ATCC). .. Each 50% synthetic variant sample was a mixture of 2 ng/μL human cell-line gDNA NA18572 (Coriell Biorepository), 50 copies/μL of wildtype Mycobacterium tuberculosis genomic DNA (ATCC), and 50 copies/μL of synthetic template.
Mutagenesis:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
DNA Extraction:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
Isolation:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
Article Title: Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine
Article Snippet: .. We used human genomic DNA isolated from GM12878 cells (NA12878, provided by Coriell Institute), K562 cells (provided by ATCC) and breast tissue (Biochain D8235086-PP-10) for RIMSseq sequencing in this study. .. We used human genomic DNA isolated from GM12878 cells (NA12878, provided by Coriell Institute), K562 cells (provided by ATCC) and breast tissue (Biochain D8235086-PP-10) for RIMSseq sequencing in this study.
Article Title: Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine.
Article Snippet: .. 345 346 Methods 347 348 RIMS-seq2 library preparation 349 We used human genomic DNA isolated from GM12878 cells (referred as NA12878, provided by Coriell 350 Institute), K562 cells (provided by ATCC) and paired breast tumor/tissue (Biochain D8235086-PP-10) for 351 RIMS-seq2 sequencing in this study. .. 345 346 Methods 347 348 RIMS-seq2 library preparation 349 We used human genomic DNA isolated from GM12878 cells (referred as NA12878, provided by Coriell 350 Institute), K562 cells (provided by ATCC) and paired breast tumor/tissue (Biochain D8235086-PP-10) for 351 RIMS-seq2 sequencing in this study.
SYBR Green Assay:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
Real-time Polymerase Chain Reaction:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
Concentration Assay:Article Title: Polynucleotide primers and probes
Article Snippet: .. KRAS G12V Mutation Assay with SybrGreen Dye Materials: 0.1× TE buffer: 10 mM Tris, 0.1 mM EDTA pH=8.0 Genomic DNA isolation kit: Qiagen DNeasy Blood & Tissue Kit #69504 Wild Type human genomic DNA template: Promega human genomic DNA #G1471 Mutant template: (G12V) genomic DNA isolated from freshly harvested SW480 colorectal adenocarcinoma cells (ATCC#CCL-228) Oligonucleotides: kras conventional forward primer 10-53 (SEQ ID NO: 6), kras G12V conventional reverse primer 10-48 (SEQ ID NO: 5), kras P10-56 (SEQ ID NO: 8) (i.e., “first polynucleotide”), kras F10-54 (SEQ ID NO: 7) (i.e., “second polynucleotide”), Real time SYBR Green qPCR mix: BioRad IQ SYBR Green Supermix #170-8882 Methods: Genomic DNA isolation: Kras G12V human genomic DNA was isolated from freshly harvested SW480 cells using Qiagen DNeasy Blood & Tissue Kit according to manufacturer protocol, resuspended in 0.1× TE buffer at a concentration of 100 ng/1.11, aliquoted and stored at −20° C. until use. .. Template preparation: To generate genomic DNA templates for real time PCR reactions, Promega WT genomic DNA was spiked with the designated amount of has G12V genomic DNA so that number of mutant has copies varied from one to 14,000 copies per 50 ng of total DNA, then template DNA was aliquoted and stored at −20° C. until use.
Sequencing:Article Title: Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine
Article Snippet: .. We used human genomic DNA isolated from GM12878 cells (NA12878, provided by Coriell Institute), K562 cells (provided by ATCC) and breast tissue (Biochain D8235086-PP-10) for RIMSseq sequencing in this study. .. We used human genomic DNA isolated from GM12878 cells (NA12878, provided by Coriell Institute), K562 cells (provided by ATCC) and breast tissue (Biochain D8235086-PP-10) for RIMSseq sequencing in this study.
Article Title: Simultaneous assessment of human genome and methylome data in a single experiment using limited deamination of methylated cytosine.
Article Snippet: .. 345 346 Methods 347 348 RIMS-seq2 library preparation 349 We used human genomic DNA isolated from GM12878 cells (referred as NA12878, provided by Coriell 350 Institute), K562 cells (provided by ATCC) and paired breast tumor/tissue (Biochain D8235086-PP-10) for 351 RIMS-seq2 sequencing in this study. .. 345 346 Methods 347 348 RIMS-seq2 library preparation 349 We used human genomic DNA isolated from GM12878 cells (referred as NA12878, provided by Coriell 350 Institute), K562 cells (provided by ATCC) and paired breast tumor/tissue (Biochain D8235086-PP-10) for 351 RIMS-seq2 sequencing in this study.
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