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Accuprime gc rich dna polymerase

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AccuPrime™ GC-Rich DNA Polymerase is a thermostable DNA polymerase designed for the amplification of GC-rich DNA templates. It exhibits high fidelity and robust performance, making it suitable for a variety of PCR applications.

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Lab products found in correlation

Truseq stranded mrna sample preparation kit, by Illumina (3 mentions) Lenti pac hiv expression packaging kit, by GeneCopoeia (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Amaxa nucleofector 2, by Lonza (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Repli g, by Qiagen (2 mentions) Nimbelgen seqcap ez solution bait sets, by Roche (2 mentions) E gel, by Thermo Fisher Scientific (2 mentions) Casava 1, by Illumina (2 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Veriti thermal cycler, by Thermo Fisher Scientific (2 mentions) Miniseq sequencing system, by Illumina (1 mentions) Hotstartaq dna polymerase, by Qiagen (1 mentions) Blood and tissue kit, by Qiagen (1 mentions) Longamp taq pcr kit, by New England Biolabs (1 mentions) Abi 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions) Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions) Qiaprep spin miniprep kit, by Qiagen (1 mentions) Chromas, by Technelysium (1 mentions) Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)

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DNA polymerase (71 citations) AccuPrime DNA Polymerase (17 citations) Accuprime polymerase (15 citations) AccuPrime (15 citations)

27 protocols using accuprime gc rich dna polymerase

1

Cloning and Lentiviral Transduction of Candidate Promoters

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Candidate promoters of 200 bp were PCR amplified using AccuPrime™ GC-Rich DNA Polymerase (ThermoFisher Scientific 12337016) and cloned into the pLSmP-Luciferase vector after digestion with SbfI and AgeI restriction enzymes (remove minimal promoter). Primers with 20bp homology to the vector cloning site were designed and PCR products were assembled to the lentiviral vector using NEBuilder® HiFi DNA Assembly Master Mix (E2621S). Lentiviruses were produced using Lenti-Pac HIV Expression Packaging Kit (Genecopoeia, LT001) according to manufacturer's instructions. Small scale viral productions on HEK293T cells (2x800,000 cells seeded on a p6 well 24h prior to transfection; virus-containing culture media was collected 48h post-transfection and was used to infect desired cells) were performed of all different constructs to test luciferase activity in four different cell lines (MCF-7, IMR-90, K562, HEK293T). 50,000 cells were seeded on 96-well plates in a volume of 50 μL and another 50 μL of virus-containing medium was added in order to transduce them. Luminescence was measured 24h or 48h post-infection using Dual-Luciferase® Reporter Assay System (Promega, E1910).
Georgakopoulos-Soares I., Victorino J., Parada G.E., Agarwal V., Zhao J., Wong H.Y., Umar M.I., Elor O., Muhwezi A., An J.Y., Sanders S.J., Kwok C.K., Inoue F., Hemberg M, & Ahituv N. (2022). High-throughput characterization of the role of non-B DNA motifs on promoter function. Cell Genomics, 2(4), 100111.
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2

PHOX2B Genetic Variant Identification

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All three exons of the PHOX2B coding region were analyzed using published protocols (Bachetti et al., 2005b; Matera et al., 2004), in which their amplification was followed by product purification and direct Sanger sequencing. Mosaicism was analyzed by modifying a previously reported protocol: the region spanning the polyAla stretch was amplified using a 6′FAM‐conjugated oligonucleotide and AccuPrime GC‐Rich DNA Polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and the PCR product was run using the capillary electrophoresis method (see Bachetti, Parodi, Di Duca, Santamaria, Ravazzolo, & Ceccherini, 2011 for details).
GenBank sequence NG_008243.1 was used as reference for the PHOX2B gene while NM_003924.3 and NP_003915.2 were used as reference sequences for PHOX2B cDNA and protein respectively.
The PHOX2B variants identified in our cohort of patients have been submitted to the locus‐specific database (LSDB) at https://www.lovd.nl/PHOX2B.
Di Lascio S., Benfante R., Di Zanni E., Cardani S., Adamo A., Fornasari D., Ceccherini I, & Bachetti T. (2017). Structural and functional differences in PHOX2B frameshift mutations underlie isolated or syndromic congenital central hypoventilation syndrome. Human Mutation, 39(2), 219-236.
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3

Quantifying CRISPR-Cas9 Induced Mutations

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Indel frequencies were measured with bulk cell cultures 5 days after electroporation. Briefly, genomic DNA was extracted using the Qiagen Blood and Tissue kit. Genomic region surrounding the sgRNA targeting site was amplified using either AccuPrime GC-Rich DNA polymerase (ThermoFisher Scientific, Cat# 12337016) for sgRNA ELANE e2–71C targeting site or HotStarTaq DNA polymerase (QIAGEN, Cat# 203203) for other PCR reactions strictly following the manufactory instructions with variable annealing temperature. PCR products were subject to Sanger sequencing and then TIDE analysis to identifies the major induced mutations within 30 bp away from the projected editing site and accurately determines their frequency in a cell population(Brinkman et al., 2014 (link)). For amplicon deep sequencing, we generated amplicon libraries with 2 rounds of PCR using primers containing sample-specific barcodes and adapter. Amplicons were then sequenced for 2 × 150 bp paried-end reads with MiniSeq Sequencing system (Illumina). The deep sequencing data were analyzed by CRISPResso (Clement et al., 2019 (link)) software with the following parameters: a minimum alignment identity of 75%, window size of 2 bp around the cleavage site to quantify indels, an average PHRED quality score of 30, and excluded substitutions to limit potential false-positives.
Rao S., Yao Y., de Brito J.S., Yao Q., Shen A., Watkinson R., Kennedy A., Coyne S., Ren C., Zeng J., Serbin A.V., Studer S., Ballotti K., Harris C., Luk K., Stevens C., Armant M., Pinello L., Wolfe S.A., Chiarle R., Shimamura A., Lee B., Newburger P.E, & Bauer D.E. (2021). Dissecting ELANE neutropenia pathogenicity by human HSC gene editing. Cell stem cell, 28(5), 833-845.e5.
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4

Lentiviral Promoter Screening in Cell Lines

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Candidate promoters of 200 bp were PCR amplified using AccuPrime™ GC-Rich DNA Polymerase (ThermoFisher Scientific 12337016) and cloned into the pLSmP-Luciferase vector after digestion with SbfI and AgeI restriction enzymes (remove minimal promoter). Primers with 20bp homology to the vector cloning site were designed and PCR products were assembled to the lentiviral vector using NEBuilder® HiFi DNA Assembly Master Mix (E2621S). Lentiviruses were produced using Lenti-Pac HIV Expression Packaging Kit (Genecopoeia, LT001) according to manufacturer’s instructions. Small scale viral productions on HEK293T cells (2×800,000 cells seeded on a p6 well 24h prior to transfection; virus-containing culture media was collected 48h post-transfection and was used to infect desired cells) were performed of all different constructs to test luciferase activity in four different cell lines (MCF-7, IMR-90, K562, HEK293T). 50,000 cells were seeded on 96-well plates in a volume of 50 μL and another 50 μL of virus-containing medium was added in order to transduce them. Luminescence was measured 24h or 48h post-infection using Dual-Luciferase® Reporter Assay System (Promega, E1910).
Georgakopoulos-Soares I., Victorino J., Parada G.E., Agarwal V., Zhao J., Wong H.Y., Umar M.I., Elor O., Muhwezi A., An J.Y., Sanders S.J., Kwok C.K., Inoue F., Hemberg M, & Ahituv N. (2022). High-throughput characterization of the role of non-B DNA motifs on promoter function. Cell Genomics, 2(4), 100111.
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5

Targeted Genomic Integration in RPE1 Cells

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Generating and screening of targeted clones were performed as described38 (link), with the following modifications. 106 RPE1 cells with 2, 3, or 4 copies of chromosome 21 were nucleofected with 2 µg pAAVS1-DRGFP or pAAVS1-DRGFPCE plasmid together with 2 µg pZFN-AAVS1, using program X-001 of the Amaxa nucleofector II (Lonza). Targeting of individual clones was confirmed by PCR using the Accuprime GC-rich DNA polymerase (Life Technologies). The presence of a single integrant was determined by qPCR (data not shown).
Lane A.A., Chapuy B., Lin C.Y., Tivey T., Li H., Townsend E.C., van Bodegom D., Day T.A., Wu S.C., Liu H., Yoda A., Alexe G., Schinzel A.C., Sullivan T.J., Malinge S., Taylor J.E., Stegmaier K., Jaffe J.D., Bustin M., te Kronnie G., Izraeli S., Harris M., Stevenson K.E., Neuberg D., Silverman L.B., Sallan S.E., Bradner J.E., Hahn W.C., Crispino J.D., Pellman D, & Weinstock D.M. (2014). Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 lysine 27 trimethylation. Nature genetics, 46(6), 618-623.
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6

Primer Design and Sequencing of DLX5 and DLX6

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Primers to amplify the exons, intron/exon boundaries, and regions of high conservation within the intragenic and intergenic regions of DLX5 and DLX6 were designed in Primer3 (Table S3) [55] (link). PCR products were amplified in one CP1 NSDTR and one unaffected NSDTR. Areas with high GC content were amplified using Invitrogen AccuPrime GC-Rich DNA Polymerase protocols (Life Technologies, Grand Island, NY). PCR products were cleaned using ExoSAP-IT and sequenced using the Big Dye terminator mix on an ABI 3100 Genetic Analyzer (Applied Biosystems, CA). Sequences were analyzed using Chromas (Technelysium, Tewantin, QLD, Australia) and Vector NTI (Informax, Frederick, MD). Sequences were aligned to each other and the Boxer reference sequence (CanFam 2.0) to identify any polymorphisms [19] (link). The DLX6 LINE-1 insertion was amplified using LongAmp Taq PCR Kit (New England BioLabs Ipswich, MA) and cloned using the Invitrogen TOPO TA Cloning kit (pCR2.1-TOPO vector) with One Shot TOP10 Chemically Competent E. coli. Products were isolated with the Qiaprep Spin Miniprep kit (Qiagen, Valencia, CA) and sequenced using an ABI 3100 Genetic Analyzer (Applied Biosystems, CA). LINE element sequence was identified using BLAST 2.2.28 [23] (link).
Wolf Z.T., Leslie E.J., Arzi B., Jayashankar K., Karmi N., Jia Z., Rowland D.J., Young A., Safra N., Sliskovic S., Murray J.C., Wade C.M, & Bannasch D.L. (2014). A LINE-1 Insertion in DLX6 Is Responsible for Cleft Palate and Mandibular Abnormalities in a Canine Model of Pierre Robin Sequence. PLoS Genetics, 10(4), e1004257.
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7

Targeted Genomic Integration in RPE1 Cells

Cited 1 time
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Generating and screening of targeted clones were performed as described38 (link), with the following modifications. 106 RPE1 cells with 2, 3, or 4 copies of chromosome 21 were nucleofected with 2 µg pAAVS1-DRGFP or pAAVS1-DRGFPCE plasmid together with 2 µg pZFN-AAVS1, using program X-001 of the Amaxa nucleofector II (Lonza). Targeting of individual clones was confirmed by PCR using the Accuprime GC-rich DNA polymerase (Life Technologies). The presence of a single integrant was determined by qPCR (data not shown).
Lane A.A., Chapuy B., Lin C.Y., Tivey T., Li H., Townsend E.C., van Bodegom D., Day T.A., Wu S.C., Liu H., Yoda A., Alexe G., Schinzel A.C., Sullivan T.J., Malinge S., Taylor J.E., Stegmaier K., Jaffe J.D., Bustin M., te Kronnie G., Izraeli S., Harris M., Stevenson K.E., Neuberg D., Silverman L.B., Sallan S.E., Bradner J.E., Hahn W.C., Crispino J.D., Pellman D, & Weinstock D.M. (2014). Triplication of a 21q22 region contributes to B cell transformation through HMGN1 overexpression and loss of histone H3 lysine 27 trimethylation. Nature genetics, 46(6), 618-623.
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8

RUNX3 Exon Sequencing Protocol

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Genomic DNA was extracted using the PureLink Genomic DNA Mini Kit (Life Technologies, Thermo Fisher Scientific, Waltham, MA). All seven exons of RUNX3 were amplified using AccuPrime GC-Rich DNA Polymerase (Life Technologies). After purification, direct sequencing was performed with the BigDye A Haider et al.
Haider A., Steininger A., Ullmann R., Hummel M., Dimitrova L., Beyer M., Vandersee S., Lenze D., Sterry W., Assaf C, & Möbs M. (2016). Inactivation of RUNX3/p46 Promotes Cutaneous T-Cell Lymphoma. The Journal of investigative dermatology, 136(11).
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9

Cloning and Characterization of Prkd1 Promoter

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Genomic DNA was isolated from MN9D cells using the DNeasy Blood & Tissue Kit (Qiagen). The 1.6-kb (−1370/+250) mouse Prkd1 promoter region was amplified by PCR using mouse genomic DNA as a template and the primer set P-1370F / P+250R. PCR conditions were as follows: 95°C for 5 min; 38 cycles of 95°C for 45 sec, 61°C for 30 sec, and 68°C for 2 min; and then 68°C for 7 min. The PCR reaction was performed with the AccuPrime GC-rich DNA polymerase (Invitrogen). DMSO was added to the PCR mixture at a final concentration of 5% (v/v) to increase amplification efficiency. Following double digestion by Kpn1/Nhe1, the 1.6-kb PCR product was cloned into the pGL4.14 luciferase vector (Promega, Madison, WI) and designated as pGL4-1370/+250. Using pGL4-1370/+250 as a template, a set of 5’ and 3’ end-truncated Prkd1 promoter constructs were created by PCR with appropriate primers (Table 1) and then cloned into the pGL4.14 luciferase vector similar to the preparation of pGL4-1370/+250. All reporter constructs were verified by DNA sequencing.
Ay M., Jin H., Harischandra D.S., Asaithambi A., Kanthasamy A., Anantharam V, & Kanthasamy A.G. (2015). Molecular Cloning, Epigenetic Regulation and Functional Characterization of Prkd1 Gene Promoter in Dopaminergic Cell Culture Models of Parkinson’s Disease. Journal of neurochemistry, 135(2), 402-415.
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10

Telomere TELVI-R Amplification and Sequencing

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The telomere TELVI-R was amplified by PCR using the AccuPrime™ GC-Rich DNA Polymerase (InVitrogen) and the TELVI-R specific primer 5′-CGTGTGCGTACGCCATATCAATATG-3′ and cloned into TA-cloning vector (InVitrogen)24 (link). Sequencing was perfomed by GATC BIOTECH. Telomere sequences were aligned using EMBOSS needle website. Conserved sequences were defined when perfectly matching to the consensus and upon single point mutation or 1–2-bp deletion/insertions. The point of divergence was defined when the sequence could not be aligned with the reference sequence.
Aguilera P., Whalen J., Minguet C., Churikov D., Freudenreich C., Simon M.N, & Géli V. (2020). The nuclear pore complex prevents sister chromatid recombination during replicative senescence. Nature Communications, 11, 160.
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