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Q5 high fidelity polymerase

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The Q5 High-Fidelity Polymerase is a DNA polymerase enzyme that can be used for high-fidelity DNA amplification. It possesses 3'→5' exonuclease activity, which provides a proofreading function to ensure accurate DNA synthesis.

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Lab products found in correlation

300 cycle v2 kit, by Illumina (2 mentions) Miseq sequencer, by Illumina (2 mentions) Qiaquick pcr purification kit, by Qiagen (1 mentions) Miseq machine, by Illumina (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions) Q5 high fidelity polymerase, by Qiagen (1 mentions)

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Q5 polymerase (4 citations)

3 protocols using q5 high fidelity polymerase

1

Next-Generation Sequencing of Gene Editing

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Gene editing efficiencies were assessed by next-generation sequencing (NGS) using a two-step PCR-based method. Briefly, genomic DNA from cells was extracted with the DNeasy Blood and Tissue Kit (QIAGEN) and amplified for first-step PCR with Q5 High-Fidelity Polymerase (NEB). Target sequences in EMX1 and DYRK1A loci and primer sequences carrying 5′ Illumina sequencing adaptors for PCR amplification are provided in Supplementary Table S2. PCR products were purified using QIAquick PCR Purification Kit (Qiagen) and serve as template for second-step PCR with primer sequences carrying Illumina barcodes by Q5 High-Fidelity Polymerase (NEB). The PCR products were sequenced on an Illumina MiSeq machine by PE150 via commercial sequencing service (Tsingke Biotechnology) and the efficiency of genome editing was determined from the sequencing data using CRISPResso2 (43 (link)).
Song G., Zhang F., Tian C., Gao X., Zhu X., Fan D, & Tian Y. (2022). Discovery of potent and versatile CRISPR–Cas9 inhibitors engineered for chemically controllable genome editing. Nucleic Acids Research, 50(5), 2836-2853.
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2

Next-Generation Sequencing of Genome Modifications

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Genome modification was measured by next-generation sequencing using a 2-step PCR-based Illumina library construction method. Briefly, genomic regions were initially amplified using Q5 High-Fidelity DNA Polymerase (NEB), human cell lysate containing ~100 ng of genomic DNA, and gene-specific round 1 primers (Supplementary Table 4c). PCR products were purified using paramagnetic beads as previously described41 and diluted 1:100 to serve as template for a second round of PCR using Q5 High-Fidelity Polymerase and primers encoding Illumina barcodes and adapter sequences (Supplementary Table 4c). PCR products were purified prior to quantification (via Qiagen QIAxcel electrophoresis), normalization, and pooling. Final libraries were quantified by qPCR (Illumina Library qPCR Quantification Kit, KAPA Biosystems) and sequenced on a MiSeq sequencer using a 300-cycle v2 kit (Illumina). Genome editing activities were determined from the sequencing data using CRISPResso2 (Clement et al., 2019) with commands --min_reads_to_use_region 100, -w 10, and for certain sequencing data sets --ignore_substitutions.
Mahendra C., Christie K.A., Osuna B.A., Pinilla-Redondo R., Kleinstiver B.P, & Bondy-Denomy J. (2020). Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nature microbiology, 5(4), 620-629.
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3

Next-Generation Sequencing of Genome Modifications

Check if the same lab product or an alternative is used in the 5 most similar protocols
Genome modification was measured by next-generation sequencing using a 2-step PCR-based Illumina library construction method. Briefly, genomic regions were initially amplified using Q5 High-Fidelity DNA Polymerase (NEB), human cell lysate containing ~100 ng of genomic DNA, and gene-specific round 1 primers (Supplementary Table 4c). PCR products were purified using paramagnetic beads as previously described41 and diluted 1:100 to serve as template for a second round of PCR using Q5 High-Fidelity Polymerase and primers encoding Illumina barcodes and adapter sequences (Supplementary Table 4c). PCR products were purified prior to quantification (via Qiagen QIAxcel electrophoresis), normalization, and pooling. Final libraries were quantified by qPCR (Illumina Library qPCR Quantification Kit, KAPA Biosystems) and sequenced on a MiSeq sequencer using a 300-cycle v2 kit (Illumina). Genome editing activities were determined from the sequencing data using CRISPResso2 (Clement et al., 2019) with commands --min_reads_to_use_region 100, -w 10, and for certain sequencing data sets --ignore_substitutions.
Mahendra C., Christie K.A., Osuna B.A., Pinilla-Redondo R., Kleinstiver B.P, & Bondy-Denomy J. (2020). Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nature microbiology, 5(4), 620-629.
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