Structured Review

Roche gc rich solution
Bisulfite footprinting by deep-sequencing across the <t>FMR1</t> repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate <t>(G-rich;</t> left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
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Images

1) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

Journal: Genetics

doi: 10.1534/genetics.118.301672

Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

Techniques Used: Footprinting, Sequencing, Amplification, Methylation

Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

Techniques Used: Synthesized, Mutagenesis

2) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

Journal: Genetics

doi: 10.1534/genetics.118.301672

Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

Techniques Used: Footprinting, Sequencing, Amplification, Methylation

Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

Techniques Used: Synthesized, Mutagenesis

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Diagnostic Assay:

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Clone Assay:

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Amplification:

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Filtration:

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
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Positive Control:

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: Bisulfite-modified DNA extracted from leukocytes of a healthy individual served as a positive control for the unmethylated alleles, and water was used as a negative control. .. Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany).

Synthesized:

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: In brief, 124 base single-stranded DNA containing G4 C2 , C4 G2 , or A4 C2 hexanucleotide repeats with restriction enzyme sites (NheI or HindIII) were synthesized (Suppl. .. 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen).

Construct:

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen). .. The DNA sequence of all constructs was verified.

Electrophoresis:

Article Title: Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints
Article Snippet: PCRs were done in a reaction mixture containing 0.1 µg template DNA, 3 µl GC-rich solution, 1.5 µl 10× buffer containing MgCl2 , 2.4 µl 10 mM dNTP's, 0.6 µl each primer (5 pmol/µl) and 0.12 µl FastStart Taq (Roche, Germany) made up to 15 µl with H2 O. Amplification comprised an initial 6 min template denaturation followed by 35 cycles using the appropriate annealing temperature (listed in ) and an extension time of 30 s to 1 min 30 s depending on the length of the amplicon. .. PCR product was checked by electrophoresis through an agarose gel and an aliquot was treated with ExoSAP-IT (USB).

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. PCR was run at 96°C for 7 min followed by 35 cycles at 94°C for 30 s, 64°C for 30 s and 72°C for 1 min. PCR products were excised from 3% agarose gels (Seakem, FMC Bioproducts, Rockland, ME, USA) stained with ethidium bromide (0.56 mg/l gel, Sigma Chemicals, St. Louis, MO, USA) following high-voltage electrophoresis and purified using Qiaquick gel extraction kit (Qiagen).

Incubation:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Diagnostic Amplification & Identification Initial virus genetic detection and analysis was conducted on total RNA extracted from infected Vero E6 cells, using Tripure Isolation Reagent (Roche Applied Science, Indianapolis, IN) in a ratio of 1∶5 and incubated at room temperature for a minimum of 10 min. Total RNA was isolated by using the RNaid Kit following the manufacturer's recommendations (Qbiogene Inc., Carlsbad, CA), and the extracted RNA was reconstituted in 50 µL H2 O. .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C.

Activity Assay:

Article Title: Transmission of Intestinal Bifidobacterium longum subsp. longum Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ †
Article Snippet: The selected genes encode the following proteins: class III stress response-related ATPase with chaperone activity ( clpC ), DNA primase ( dnaG ), chaperone protein DnaJ ( dnaJ ), GTP-binding protein chain elongation factor G ( fusA ), the β subunit of DNA gyrase ( gyrB ), amidophosphoribosyltransferase ( purF ), and the β subunit of RNA polymerase ( rpoB ) ( ). .. Each 25-μl PCR mixture contained 1× PCR buffer, 200 μM deoxynucleoside triphosphates, 2 mM MgCl2 , 0.4 μM each primer, 10 μl GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA.

Modification:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: Paragraph title: Polymerase chain reaction amplification of modified DNAs ... For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche).

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Article Snippet: Native genomic DNA (1 µg) was modified by bisulfite treatment (EZ DNA methylation Kit; Zymo Research), with a slight modification. .. For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: DNA extracted from the leukocytes of healthy individuals and treated with Sss I methyltransferase (New England Biolabs, Beverly, MA, United States) prior to bisulfite modification was used as a positive control for the methylated alleles. .. Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany).

Article Title: Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota
Article Snippet: Multilocus sequencing typing (MLST) analysis For the species belonging to Bifidobacterium adolescentis , Bifidobacterium bifidum , B. catenulatum , and B. pseudocatenulatum isolates, multilocus sequencing typing (MLST) analysis was performed using the seven housekeeping genes (clpC , fusA , gyrB , ileS , purF , rplB , rpoB ) previously described in the MLST protocol by Delétoile et al. , with slight modification. .. Each 25-µL reaction contained 1× PCR buffer, 200 µM dNTPs, 2 mM MgCl2 , 0.4 µM of each primer , 10 µL GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA.

DNA Synthesis:

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: Paragraph title: DNA synthesis and plasmid construction for in vitro transcription ... 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen).

Footprinting:

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Article Snippet: Paragraph title: Colony bisulfite footprinting analysis ... For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

Infection:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Diagnostic Amplification & Identification Initial virus genetic detection and analysis was conducted on total RNA extracted from infected Vero E6 cells, using Tripure Isolation Reagent (Roche Applied Science, Indianapolis, IN) in a ratio of 1∶5 and incubated at room temperature for a minimum of 10 min. Total RNA was isolated by using the RNaid Kit following the manufacturer's recommendations (Qbiogene Inc., Carlsbad, CA), and the extracted RNA was reconstituted in 50 µL H2 O. .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C.

DNA Sequencing:

Article Title: The isolation and characterisation of the wheat molecular ZIPper I homologue, TaZYP1
Article Snippet: Each PCR contained 100 ng cDNA, 0.2 mM dNTPs, 0.2 μM primers, 1U FastStart high fidelity Taq polymerase (Roche Applied Science, Mannheim, Germany) in 25 μL of 1× high fidelity buffer supplemented with 1× GC-RICH solution (Roche). .. PCR products were cloned into pCR8/GW/TOPO (Invitrogen) for DNA sequencing (15× coverage).

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). .. To verify the PCR product, the methylation-specific PCR amplicons from 5 samples were purified with HiYield PCR DNA Extraction Kit (Real Biotech, Banqiao, Taiwan) and subjected to direct DNA sequencing (ABI 3100, Applied Biosystems, Foster City, CA, United States).

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Article Snippet: Sequencing Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). .. DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: Paragraph title: Amplification of the 5'-, 3'- and coding regions of the Pk gene for DNA sequencing ... The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany).

Sequencing:

Article Title: The isolation and characterisation of the wheat molecular ZIPper I homologue, TaZYP1
Article Snippet: Paragraph title: cDNA amplification and sequencing of the ZYP1 coding sequence ... Each PCR contained 100 ng cDNA, 0.2 mM dNTPs, 0.2 μM primers, 1U FastStart high fidelity Taq polymerase (Roche Applied Science, Mannheim, Germany) in 25 μL of 1× high fidelity buffer supplemented with 1× GC-RICH solution (Roche).

Article Title: A novel PCFT gene mutation (p.Cys66LeufsX99) causing hereditary folate malabsorption
Article Snippet: The genomic DNA sequence of this gene was taken from Ensembl ( http://www.ensembl.org/index.html ) and primer pairs for the translated exons flanking exon–intron boundaries were designed using primer3 software ( http://fokker.wi.mit.edu/primer3/input.htm ). .. For amplification of difficult fragments such as GC rich sequences either 10% DMSO or GC rich solution of FastStart Taq DNA Polymerase (Roche Diagnostics Ltd., Burgess Hill, UK) was added to get PCR product.

Article Title: Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints
Article Snippet: Paragraph title: PCR and sequencing ... PCRs were done in a reaction mixture containing 0.1 µg template DNA, 3 µl GC-rich solution, 1.5 µl 10× buffer containing MgCl2 , 2.4 µl 10 mM dNTP's, 0.6 µl each primer (5 pmol/µl) and 0.12 µl FastStart Taq (Roche, Germany) made up to 15 µl with H2 O. Amplification comprised an initial 6 min template denaturation followed by 35 cycles using the appropriate annealing temperature (listed in ) and an extension time of 30 s to 1 min 30 s depending on the length of the amplicon.

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche). .. Amplified products were cloned, and single colonies were analyzed for cytosine conversions by direct sequencing (ABI 3130), using the BigDye Terminator v3.1 Cycle Sequencing Kit.

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C. .. Sequence was further analyzed using Sequencher (Gene Codes Corporation, Ann Arbor, MI).

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Article Snippet: .. Sequencing Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). .. DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).

Article Title: Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota
Article Snippet: Paragraph title: Multilocus sequencing typing (MLST) analysis ... Each 25-µL reaction contained 1× PCR buffer, 200 µM dNTPs, 2 mM MgCl2 , 0.4 µM of each primer , 10 µL GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA.

Article Title: Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men
Article Snippet: To determine the length of the CAG and GGN repeats the corresponding regions located on the exon 1 of the AR gene (Genbank accession no. M27423) were amplified using two pairs of primers whose sequence has been previously reported . .. Amplification was performed in a 25 µl reaction volume, containing 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate, 1x Fast Start Taq DNA polymerase Buffer (Roche Applied Science, Mannheim, Germany), 1x GC-rich solution buffer (Roche Applied Science) and 1U of Fast Start Taq DNA polymerase (Roche Applied Science).

Article Title: Transmission of Intestinal Bifidobacterium longum subsp. longum Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ †
Article Snippet: The DNA sequences of these candidate loci were selected based on the genome sequence data for strains Bifidobacterium longum DJO10A ( ) and Bifidobacterium longum NCC2705 ( ). .. Each 25-μl PCR mixture contained 1× PCR buffer, 200 μM deoxynucleoside triphosphates, 2 mM MgCl2 , 0.4 μM each primer, 10 μl GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA.

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen). .. The DNA sequence of all constructs was verified.

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. The Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 310 Genetic Analyser (Applied Biosystems) were used for direct DNA sequencing with capillary electrophoresis and automated fluorescence-based detection according to the manufacturer's instructions.

Cellular Antioxidant Activity Assay:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Broadly reactive Arenavirus primers used for initial identification were designed for the L polymerase gene on the L segment (L4160F, GCA GAR TTY AAA TCI AGA TT ; L4393R, CCR TYI ASC CAR TCT ITI ACA TC ; L4292F, GAT CAT TCI RTY GCI AAT GG ; L4841R, CAI AII CCT ATA AAI CCW GAT G ) and the glycoprotein gene on the S segment (GP878+, GAC RTG CCW GGI GGI TAY TG ; GP1126-, TAC CAA AAT TTG TGT ART TRC ART AIG G ; GP1153+, CCT TAY TGY AAY TAC ACI AAA TTT TGG T ; GP1396-, ATG TGY CTR TGI GTI GGI AW ). .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C.

DNA Extraction:

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). .. To verify the PCR product, the methylation-specific PCR amplicons from 5 samples were purified with HiYield PCR DNA Extraction Kit (Real Biotech, Banqiao, Taiwan) and subjected to direct DNA sequencing (ABI 3100, Applied Biosystems, Foster City, CA, United States).

Fluorescence:

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. The Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI PRISM 310 Genetic Analyser (Applied Biosystems) were used for direct DNA sequencing with capillary electrophoresis and automated fluorescence-based detection according to the manufacturer's instructions.

Methylation:

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: DNA extracted from the leukocytes of healthy individuals and treated with Sss I methyltransferase (New England Biolabs, Beverly, MA, United States) prior to bisulfite modification was used as a positive control for the methylated alleles. .. Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany).

Mutagenesis:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: Polymerase chain reaction amplification of modified DNAs pUC19-based plasmids containing intrinsically straight ∼200-bp sequences ( ) were subjected to site-directed mutagenesis to obtain suitable restriction sites ( Supplementary Data S2 ). .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche).

Isolation:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Diagnostic Amplification & Identification Initial virus genetic detection and analysis was conducted on total RNA extracted from infected Vero E6 cells, using Tripure Isolation Reagent (Roche Applied Science, Indianapolis, IN) in a ratio of 1∶5 and incubated at room temperature for a minimum of 10 min. Total RNA was isolated by using the RNaid Kit following the manufacturer's recommendations (Qbiogene Inc., Carlsbad, CA), and the extracted RNA was reconstituted in 50 µL H2 O. .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C.

Size-exclusion Chromatography:

Article Title: Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men
Article Snippet: Amplification was performed in a 25 µl reaction volume, containing 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate, 1x Fast Start Taq DNA polymerase Buffer (Roche Applied Science, Mannheim, Germany), 1x GC-rich solution buffer (Roche Applied Science) and 1U of Fast Start Taq DNA polymerase (Roche Applied Science). .. PCR conditions were: 30 cycles of 95°C for 45 sec, 56°C for 30 sec and 72°C for 30 sec for CAG amplification; 30 cycles of 95°C for 1 min, 55°C for 2 min and 72°C for 2 min for GGN amplification.

Purification:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche).

Article Title: A novel PCFT gene mutation (p.Cys66LeufsX99) causing hereditary folate malabsorption
Article Snippet: For amplification of difficult fragments such as GC rich sequences either 10% DMSO or GC rich solution of FastStart Taq DNA Polymerase (Roche Diagnostics Ltd., Burgess Hill, UK) was added to get PCR product. .. The PCR products purified with MicroCLEAN (Web Scientific, Crewe, UK) were directly sequenced by Big Dye Terminator Cycle Sequencing System (Applied Biosystem) and cleaned up using the EDTA method of precipitation.

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C. .. Resulting DNA products were visualized and purified using a 1% agarose gel, and the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA).

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). .. To verify the PCR product, the methylation-specific PCR amplicons from 5 samples were purified with HiYield PCR DNA Extraction Kit (Real Biotech, Banqiao, Taiwan) and subjected to direct DNA sequencing (ABI 3100, Applied Biosystems, Foster City, CA, United States).

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Article Snippet: .. Sequencing Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). .. DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. PCR was run at 96°C for 7 min followed by 35 cycles at 94°C for 30 s, 64°C for 30 s and 72°C for 1 min. PCR products were excised from 3% agarose gels (Seakem, FMC Bioproducts, Rockland, ME, USA) stained with ethidium bromide (0.56 mg/l gel, Sigma Chemicals, St. Louis, MO, USA) following high-voltage electrophoresis and purified using Qiaquick gel extraction kit (Qiagen).

Polymerase Chain Reaction:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche). .. To generate DNA where only one strand contained analog 8 , the previous conditions were modified so that the template was replaced with the desired amount of unmodified PCR product and the number of cycles was reduced from 30 to a single extension cycle.

Article Title: The isolation and characterisation of the wheat molecular ZIPper I homologue, TaZYP1
Article Snippet: .. Each PCR contained 100 ng cDNA, 0.2 mM dNTPs, 0.2 μM primers, 1U FastStart high fidelity Taq polymerase (Roche Applied Science, Mannheim, Germany) in 25 μL of 1× high fidelity buffer supplemented with 1× GC-RICH solution (Roche). .. PCR products were cloned into pCR8/GW/TOPO (Invitrogen) for DNA sequencing (15× coverage).

Article Title: A novel PCFT gene mutation (p.Cys66LeufsX99) causing hereditary folate malabsorption
Article Snippet: .. For amplification of difficult fragments such as GC rich sequences either 10% DMSO or GC rich solution of FastStart Taq DNA Polymerase (Roche Diagnostics Ltd., Burgess Hill, UK) was added to get PCR product. .. PCR conditions were an initial denaturation step at 95 °C for 5 min followed 35 cycles 30 s denaturation at 95 °C, 1 min annealing at 57–61.5 °C (depending on fragment) and 1 min extension at 72 °C with a final extension at 72 °C for 5 min.

Article Title: Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints
Article Snippet: Paragraph title: PCR and sequencing ... PCRs were done in a reaction mixture containing 0.1 µg template DNA, 3 µl GC-rich solution, 1.5 µl 10× buffer containing MgCl2 , 2.4 µl 10 mM dNTP's, 0.6 µl each primer (5 pmol/µl) and 0.12 µl FastStart Taq (Roche, Germany) made up to 15 µl with H2 O. Amplification comprised an initial 6 min template denaturation followed by 35 cycles using the appropriate annealing temperature (listed in ) and an extension time of 30 s to 1 min 30 s depending on the length of the amplicon.

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Article Snippet: .. For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche). .. Amplified products were cloned, and single colonies were analyzed for cytosine conversions by direct sequencing (ABI 3130), using the BigDye Terminator v3.1 Cycle Sequencing Kit.

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C. .. Resulting DNA products were visualized and purified using a 1% agarose gel, and the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA).

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: .. Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany). .. PCR was performed in a TaKaRa PCR Thermal Cycler Dice Gradient (Otsu, Japan) for methylated RUNX3 with RUNX3 -5M, the sense primer (5’-TTACGAGGGGCGGTCGTACGCGGG-3’), and RUNX3 -3M, the antisense primer (5’-AAAACGACCGACGCGAACGCCTCC-3’), using an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 69.1 °C for 1 min, 74 °C for 1 min and a final extension at 74 °C for 7 min. For unmethylated RUNX3 , PCR was performed using RUNX3 -5U, the sense primer (5’-TTATGAGGGGTGGTTGTATGTGGG-3’) and RUNX3 -3U, the antisense primer (5’-AAAACAACCAACACAAACACCTCC-3’) following an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 1 min, 61.8 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 7 min.

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Article Snippet: .. Sequencing Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). .. DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).

Article Title: Loss of function of Ywhah in mice induces deafness and cochlear outer hair cells' degeneration
Article Snippet: A hundred nanograms of DNA was amplified with Taq DNA polymerase (AmpliTaq Gold, Applied BioSystems, Foster City, CA, USA) helped with GC-RICH resolution solution (Roche Diagnostics) for exon 1, using intronic primers representing each exon ( ). .. Standard cycling conditions were carried out with optimized annealing temperatures: 30 s at 95 °C, 1 min at 65 °C for exon 1 and 30 s at 60 °C for exon 2, and 1 min at 72 °C for the 35 cycles, followed by a final elongation at 72 °C for 5 min. PCR products were cycled-sequenced using BigDye terminator system (Applied BioSystems), and analyzed on an ABI Prism 3130 capillary sequencer (Applied BioSystems).

Article Title: Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota
Article Snippet: .. Each 25-µL reaction contained 1× PCR buffer, 200 µM dNTPs, 2 mM MgCl2 , 0.4 µM of each primer , 10 µL GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA. .. The PCR amplification program consisted of an initial heating step at 95°C for 5 min; 30 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 1 min; and a final extension step at 72°C for 10 min. For compensating unavailable nucleotide sequence for rplB gene, the following primer set was used; forward: 5′-TGGTGCTCAGGCTGATATCA-3′ , reverse: 5′ -TAAGCGCCATCCTTGGCG-3′ .

Article Title: Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men
Article Snippet: CAG and GGN repeat polymorphisms DNA was extracted from blood samples (200 µl) using High Pure PCR Template Preparation Kits (Roche Applied Science). .. Amplification was performed in a 25 µl reaction volume, containing 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate, 1x Fast Start Taq DNA polymerase Buffer (Roche Applied Science, Mannheim, Germany), 1x GC-rich solution buffer (Roche Applied Science) and 1U of Fast Start Taq DNA polymerase (Roche Applied Science).

Article Title: Transmission of Intestinal Bifidobacterium longum subsp. longum Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ Strains from Mother to Infant, Determined by Multilocus Sequencing Typing and Amplified Fragment Length Polymorphism ▿ †
Article Snippet: .. Each 25-μl PCR mixture contained 1× PCR buffer, 200 μM deoxynucleoside triphosphates, 2 mM MgCl2 , 0.4 μM each primer, 10 μl GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA. .. The PCR amplification program consisted of an initial heating step at 95°C for 5 min, 30 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 1 min, and a final extension step at 72°C for 10 min. Sequencing was performed as described above.

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: .. 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen). ..

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: .. The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. Thermocycling was undertaken in GeneAmp PCR system 2400/2700 (Perkin-Elmer/Cetus): Initial denaturation at 96°C for 7 min followed by 10 cycles at 94°C for 30 s, 62°C for 30 s and 72°C for 1 min and then 25 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. For the 3'-region of the P k gene 5 pmol of primers Pk-(1006)-F and Pk-(1881)-R were mixed with 100 ng of genomic DNA, 2 nmol of each dNTP, 2% glycerol, 1% cresol red and 0.5 U of AmpliTaq Gold (Perkin Elmer/Roche Molecular Systems) in 10 × PCR buffer with 15 mM MgCl2 .

Article Title: Isolation of the Genome Sequence Strain Mycobacterium avium 104 from Multiple Patients over a 17-Year Period
Article Snippet: .. Except for HSD, the PCR was performed by combining 5 μl of 5× GC-rich PCR buffer, 2 μl of 10 mM nucleotides, 1.5 μl of 10 mM primer mix, 2.5 μl of GC-rich resolution solution (Roche Diagnostics, Basel, Switzerland), 1 μl of 10 μg/ml template, 1 U of Taq enzyme, and sterile water to make 25 μl per reaction mixture. .. The HSD PCR mixture required the addition of 0.6 μl of 2.5 mM MgCl2 , which lowered the GC-rich resolution solution to 2 μl per reaction.

Gel Extraction:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C. .. Resulting DNA products were visualized and purified using a 1% agarose gel, and the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA).

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. PCR was run at 96°C for 7 min followed by 35 cycles at 94°C for 30 s, 64°C for 30 s and 72°C for 1 min. PCR products were excised from 3% agarose gels (Seakem, FMC Bioproducts, Rockland, ME, USA) stained with ethidium bromide (0.56 mg/l gel, Sigma Chemicals, St. Louis, MO, USA) following high-voltage electrophoresis and purified using Qiaquick gel extraction kit (Qiagen).

Chloramphenicol Acetyltransferase Assay:

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Broadly reactive Arenavirus primers used for initial identification were designed for the L polymerase gene on the L segment (L4160F, GCA GAR TTY AAA TCI AGA TT ; L4393R, CCR TYI ASC CAR TCT ITI ACA TC ; L4292F, GAT CAT TCI RTY GCI AAT GG ; L4841R, CAI AII CCT ATA AAI CCW GAT G ) and the glycoprotein gene on the S segment (GP878+, GAC RTG CCW GGI GGI TAY TG ; GP1126-, TAC CAA AAT TTG TGT ART TRC ART AIG G ; GP1153+, CCT TAY TGY AAY TAC ACI AAA TTT TGG T ; GP1396-, ATG TGY CTR TGI GTI GGI AW ). .. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C.

Plasmid Preparation:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: For analog 2 , PCR reactions (100 µl) included 20 ng plasmid template (or alternatively 20 ng of purified PCR product from a previous reaction), 0.4 mM forward and reverse primers, 100 mg/ml bovine serum albumin (BSA), Taq DNA polymerase buffer (Invitrogen), 2 mM MgCl , 0.2 mM each dNTP (with dTTP completely replaced by the analog triphosphate) and 5 U Taq DNA polymerase (Invitrogen). .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche).

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: .. 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen). ..

Software:

Article Title: A novel PCFT gene mutation (p.Cys66LeufsX99) causing hereditary folate malabsorption
Article Snippet: The genomic DNA sequence of this gene was taken from Ensembl ( http://www.ensembl.org/index.html ) and primer pairs for the translated exons flanking exon–intron boundaries were designed using primer3 software ( http://fokker.wi.mit.edu/primer3/input.htm ). .. For amplification of difficult fragments such as GC rich sequences either 10% DMSO or GC rich solution of FastStart Taq DNA Polymerase (Roche Diagnostics Ltd., Burgess Hill, UK) was added to get PCR product.

Article Title: The Scope for Thalassemia Gene Therapy by Disruption of Aberrant Regulatory Elements
Article Snippet: Sequencing Purified plasmids and PCR products were sequenced using the BigDye Terminator v1.1 Cycler sequencing kit (Applied Biosystems, Foster City, CA, USA), including 1 × GC-RICH solution (Roche, Basel, Switzerland). .. DNA sequencing products were purified using Performa® DTR Gel Filtration Cartridges Performa® DTR Gel Filtration Cartridges (Edge Biosystems, Gaithersburg, MD, USA, USA) and analyzed on a Hitachi 3031xl Genetic Analyzer with the Sequence Detection Software version 5.2 (Applied Biosystems, Foster City, MA, USA).

Article Title: Mother-to-Infant Transmission of Intestinal Bifidobacterial Strains Has an Impact on the Early Development of Vaginally Delivered Infant's Microbiota
Article Snippet: Each 25-µL reaction contained 1× PCR buffer, 200 µM dNTPs, 2 mM MgCl2 , 0.4 µM of each primer , 10 µL GC-RICH solution, 2 U Fast Taq polymerase (Roche, Basel, Switzerland), and 10 ng template DNA. .. BioNumerics software version 6.6 (Applied-Maths, Sint-Martens-Latem, Belgium) was used to perform all phylogenetic analyses.

Negative Control:

Article Title: Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue
Article Snippet: Bisulfite-modified DNA extracted from leukocytes of a healthy individual served as a positive control for the unmethylated alleles, and water was used as a negative control. .. Amplification was carried out in a 20 μL reaction volume containing 10 ng of bisulfite-modified DNA and 2 μL of 10 × PCR buffer with 20 mmol/L MgCl2 , 4 μL of a GC-rich solution, 5 pmol of each primer for RUNX3 , 200 μmol/L aliquots of each dNTP, and 1 unit of Faststart Taq DNA polymerase (Roche Applied Science, Mannheim, Germany).

Agarose Gel Electrophoresis:

Article Title: Comparative Analysis of Mycobacterium tuberculosis pe and ppe Genes Reveals High Sequence Variation and an Apparent Absence of Selective Constraints
Article Snippet: PCRs were done in a reaction mixture containing 0.1 µg template DNA, 3 µl GC-rich solution, 1.5 µl 10× buffer containing MgCl2 , 2.4 µl 10 mM dNTP's, 0.6 µl each primer (5 pmol/µl) and 0.12 µl FastStart Taq (Roche, Germany) made up to 15 µl with H2 O. Amplification comprised an initial 6 min template denaturation followed by 35 cycles using the appropriate annealing temperature (listed in ) and an extension time of 30 s to 1 min 30 s depending on the length of the amplicon. .. PCR product was checked by electrophoresis through an agarose gel and an aliquot was treated with ExoSAP-IT (USB).

Article Title: Chapare Virus, a Newly Discovered Arenavirus Isolated from a Fatal Hemorrhagic Fever Case in Bolivia
Article Snippet: Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, (36 cycles of 1 min at 95°C, 1 min at 45°C, 2 min at 72°C), and a final elongation of 10 min at 72°C. .. Resulting DNA products were visualized and purified using a 1% agarose gel, and the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA).

In Vitro:

Article Title: Poly-glycine–alanine exacerbates C9orf72 repeat expansion-mediated DNA damage via sequestration of phosphorylated ATM and loss of nuclear hnRNPA3
Article Snippet: Paragraph title: DNA synthesis and plasmid construction for in vitro transcription ... 100 μM of complementary DNA strands were annealed in the presence of 10% GC-RICH solution (Roche) and GC-RICH PCR Reaction buffer (Roche) and cloned into pcDNA3.1(+) vector (Invitrogen).

DNA Methylation Assay:

Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA
Article Snippet: Native genomic DNA (1 µg) was modified by bisulfite treatment (EZ DNA methylation Kit; Zymo Research), with a slight modification. .. For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

Concentration Assay:

Article Title: Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men
Article Snippet: Amplification was performed in a 25 µl reaction volume, containing 50 ng of genomic DNA, 200 µM of each deoxynucleotide triphosphate, 1x Fast Start Taq DNA polymerase Buffer (Roche Applied Science, Mannheim, Germany), 1x GC-rich solution buffer (Roche Applied Science) and 1U of Fast Start Taq DNA polymerase (Roche Applied Science). .. The concentration of each pair of primers was 1.2 and 1.5 µM for the amplification of the CAG and GGN repeats, respectively.

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: .. The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. Thermocycling was undertaken in GeneAmp PCR system 2400/2700 (Perkin-Elmer/Cetus): Initial denaturation at 96°C for 7 min followed by 10 cycles at 94°C for 30 s, 62°C for 30 s and 72°C for 1 min and then 25 cycles at 94°C for 30 s, 60°C for 30 s and 72°C for 1 min. For the 3'-region of the P k gene 5 pmol of primers Pk-(1006)-F and Pk-(1881)-R were mixed with 100 ng of genomic DNA, 2 nmol of each dNTP, 2% glycerol, 1% cresol red and 0.5 U of AmpliTaq Gold (Perkin Elmer/Roche Molecular Systems) in 10 × PCR buffer with 15 mM MgCl2 .

Staining:

Article Title: Two previously proposed P1/P2-differentiating and nine novel polymorphisms at the A4GALT (Pk) locus do not correlate with the presence of the P1 blood group antigen
Article Snippet: The 5'-regulatory region of the P k gene was amplified with primers Pk-5'-(-1056)-F and Pk-int1-160-R and Pk-5'-(-131)-F and Pk-int1-160-R. Amplification was performed in a reaction volume of 22 μL with four pmol of each primer, 2 nmol of each dNTP, 100 ng of genomic DNA, GC-rich enzyme mix (0.5 U per reaction), GC-rich resolution solution and buffer with a final MgCl2 concentration of 1.5 mM (GC-rich PCR System, Roche Diagnostics GmbH, Mannheim, Germany). .. PCR was run at 96°C for 7 min followed by 35 cycles at 94°C for 30 s, 64°C for 30 s and 72°C for 1 min. PCR products were excised from 3% agarose gels (Seakem, FMC Bioproducts, Rockland, ME, USA) stained with ethidium bromide (0.56 mg/l gel, Sigma Chemicals, St. Louis, MO, USA) following high-voltage electrophoresis and purified using Qiaquick gel extraction kit (Qiagen).

Variant Assay:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara). .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche).

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    Roche gc rich solution
    Bisulfite footprinting by deep-sequencing across the <t>FMR1</t> repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate <t>(G-rich;</t> left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Gc Rich Solution, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gc rich solution/product/Roche
    Average 99 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    gc rich solution - by Bioz Stars, 2020-02
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    77
    Roche gc rich solution buffer
    Bisulfite footprinting by deep-sequencing across the <t>FMR1</t> repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate <t>(G-rich;</t> left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Gc Rich Solution Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gc rich solution buffer/product/Roche
    Average 77 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gc rich solution buffer - by Bioz Stars, 2020-02
    77/100 stars
      Buy from Supplier

    Image Search Results


    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Journal: Genetics

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    doi: 10.1534/genetics.118.301672

    Figure Lengend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

    Techniques: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Journal: Genetics

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    doi: 10.1534/genetics.118.301672

    Figure Lengend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Article Snippet: For ssDNA displacement at FMR1 , GC-RICH solution was added to the PCR mix, according to the manufacturer’s instructions (Roche).

    Techniques: Synthesized, Mutagenesis