wild type cas9 expression plasmid  (New England Biolabs)


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    Name:
    USER Enzyme
    Description:
    USER Enzyme 250 units
    Catalog Number:
    m5505l
    Price:
    297
    Size:
    250 units
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    Other Enzymes
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    New England Biolabs wild type cas9 expression plasmid
    USER Enzyme
    USER Enzyme 250 units
    https://www.bioz.com/result/wild type cas9 expression plasmid/product/New England Biolabs
    Average 95 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    wild type cas9 expression plasmid - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity"

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    Journal: Nature chemical biology

    doi: 10.1038/nchembio.1793

    Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are
    Figure Legend Snippet: Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Techniques Used: Modification, High Throughput Screening Assay, DNA Sequencing, Amplification, Transfection, Expressing, Plasmid Preparation

    Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.
    Figure Legend Snippet: Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Techniques Used: Binding Assay, Activity Assay, Standard Deviation

    2) Product Images from "A versatile element for gene addition in bacterial chromosomes"

    Article Title: A versatile element for gene addition in bacterial chromosomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1085

    Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).
    Figure Legend Snippet: Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Techniques Used: Clone Assay, Sequencing, Construct

    3) Product Images from "Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach"

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007029

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.
    Figure Legend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Techniques Used: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing

    4) Product Images from "Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications"

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dsw038

    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.
    Figure Legend Snippet: Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Techniques Used: Amplification, Construct

    5) Product Images from "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage"

    Article Title: Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

    Journal: Nature

    doi: 10.1038/nature17946

    Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .
    Figure Legend Snippet: Effects of deaminase, linker length, and linker composition on base editing a , Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, hAPOBEC3G, rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS) 3 -dCas9, and dCas9-(GGS) 3 -rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b , Coomassie-stained denaturing PAGE of the expressed and purified proteins used in (c), (d), (e), and (f). c-f , Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS) 3 (d), XTEN (e), or (GGS) 7 (f). Following incubation of 1.85 μM deaminase-dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1 .

    Techniques Used: Activity Assay, Derivative Assay, In Vitro, Incubation, Positive Control, Sequencing, Staining, Polyacrylamide Gel Electrophoresis, Purification, Isolation

    Related Articles

    Clone Assay:

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: .. CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB). .. The resulting constructs were transformed into yeast ( Saccharomyces cerevisiae ) strain BY4741.

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: Wolbachia genes encoding for putative T4SS-secreted proteins were cloned into the galactose-inducible yeast expression vector, pYES2/NT A via the USER (uracil-specific excision reagent) cloning system developed at NEB. .. PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest.

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system
    Article Snippet: Paragraph title: Cloning glycogen synthase (GYS) 3' UTR ... The GYS PCR primers contained USER enzyme (NEB) cleavage sites.

    Amplification:

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: .. PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest. .. In order to utilize the URA3 + pGO45 and pMM134 plasmids, which express GFP-CPS or Sna3p-GFP, respectively [ , ], for yeast endosome:vacuole transport studies in the presence of another URA3 + yeast expression plasmid harboring the putative w Bm effector, pGO45 and pMM134 were first converted from URA3 + to LYS2 + via homologous recombination methods by digesting pM2660 (ATCC) with HindIII [ ], and transforming yeast strains harboring either pGO45 or pMM134 with this digest.

    Synthesized:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Second strand cDNA was synthesized using the NEBNext mRNA second strand synthesis module (New England Biolabs) using the dNTP-free reaction buffer and a dTTP-free dNTP mix containing dUTP (U.S. Biochemical). .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Immediately after, a second strand cDNA was synthesized using DNA polymerase I and RNase H. After adenylation reaction of 3′ ends of DNA fragments, NEBNext Adaptor containing hairpin loop structure were ligated for hybridization. .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). .. Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV reverse transcriptase. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H−). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: First-strand cDNA was synthesized using a random hexamer primer and reverse transcriptase. .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: Synthesis of first strand cDNA was performed using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H− ), subsequently second strand cDNA was synthesized using DNA polymerase I and RNase H. After terminal repair of the sequences, the 3′ ends were adenylated and NEBnext adapters (NEB, MA, USA) were ligated. .. To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Construct:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Sequencing libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA). .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB). .. The resulting constructs were transformed into yeast ( Saccharomyces cerevisiae ) strain BY4741.

    Real-time Polymerase Chain Reaction:

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. Finally, the PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 and by qPCR.

    Incubation:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C. .. Then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: .. Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C. .. PCR was then performed using the Phusion High-Fidelity DNA polymerase (Thermo Scientific), Universal PCR primers (New England BioLabs), and the Index (X) Primer (New England BioLabs).

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. PCR was then performed with Phusion high-fidelity DNA polymerase, universal PCR primers and the index (X) primer.

    Expressing:

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: Paragraph title: Expression of CYP76AA25 and CYP750B1 in Yeast ... CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB).

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: Briefly, gene-specific PCR primers were designed to clone the predicted w Bm open reading frame into the vector expressing an amino-terminal Xpress epitope tag; these primers ( ) contained a linker complementary to pYES2/NT A, with an additional uracil, and were used to amplify the target gene with PfuTurbo Cx hotstart DNA polymerase (Stratagene). .. PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest.

    Transformation Assay:

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB). .. The resulting constructs were transformed into yeast ( Saccharomyces cerevisiae ) strain BY4741.

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system
    Article Snippet: The GYS PCR primers contained USER enzyme (NEB) cleavage sites. .. RT-PCR products were cleaved with USER enzyme (NEB), mixed with pNEB206A USER vector and transformed into competent E. coli strain NEB5alpha (NEB).

    Hybridization:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Immediately after, a second strand cDNA was synthesized using DNA polymerase I and RNase H. After adenylation reaction of 3′ ends of DNA fragments, NEBNext Adaptor containing hairpin loop structure were ligated for hybridization. .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. .. Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: After adenylation of the 3′ ends of the DNA fragments, NEBNext adaptors with a hairpin loop structure were ligated to prepare for hybridization. .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: After adenylation of the 3' ends of the DNA fragments, NEBNext adaptors with a hairpin loop structure were ligated to prepare each sample for hybridization. .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Ligation:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Double-stranded cDNA was purified with AMPure XP beads and applied to the standard Illumina library protocol including end repair, dA tailing, and ligation with Illumina sequencing adapters. .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Introduce:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs). .. Following PCR enrichment, libraries underwent fragment size confirmation using an HS DNA chip on an Agilent BioAnalyzer and were quantified using the Illumina library quantification kit (KAPA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system
    Article Snippet: Cloning glycogen synthase (GYS) 3' UTR GYS 3'UTR was cloned by from HEK293-A7 cell polyA+ RNA by RT-PCR using the Protoscript First Strand cDNA Synthesis kit (NEB) and 2× Taq mix (NEB). .. The GYS PCR primers contained USER enzyme (NEB) cleavage sites.

    Generated:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C. .. Finally, PCR products were purified by AMPure XP system and the Agilent Bioanalyzer 2100 system was used to assess sequencing library quality and the clusters were sequenced on an Illumina Hiseq 2000 platform and paired-end reads were generated.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, United States) following the manufacturer’s recommendations and index codes were added to attribute sequences. .. Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C.

    Sequencing:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Paragraph title: Temperature Shift, RNA Isolation, Primer Extension, and RNA Sequence (RNA-Seq) Analyses ... Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Paragraph title: 2.2. RNA Extraction, Library Preparation, Sequencing and De Novo Assembly ... The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: Paragraph title: RNA isolation, library construction and sequencing ... Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: Paragraph title: Library preparation for transcriptome sequencing ... Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, United States) following the manufacturer’s recommendations and index codes were added to attribute sequences. .. Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C.

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: Paragraph title: RNA isolation, cDNA library construction, and sequencing ... Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: Paragraph title: LncRNA library construction and sequencing ... Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: The NEBNext Ultra RNA Library Prep Kit for Illumina sequencing (NEB, MA, USA) following the protocol as provided by the manufacturer was used for library preparation, index sequences were added to trace back the sequences to each sample. .. To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Recombinant:

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB). .. Proteins were expressed, and microsomal fractions containing the recombinant protein were isolated as described previously ( ; ; ).

    Nucleic Acid Electrophoresis:

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: The quantity and quality of total RNA were analyzed using a spectrophotometer (Nanodrop) and by gel electrophoresis. .. Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    RNA Sequencing Assay:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Paragraph title: Temperature Shift, RNA Isolation, Primer Extension, and RNA Sequence (RNA-Seq) Analyses ... Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: Integrative Analyses of Long Non-coding RNA and mRNA Involved in Piglet Ileum Immune Response to Clostridium perfringens Type C Infection
    Article Snippet: Paragraph title: Library Preparation and RNA-Seq Data Acquisition ... Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR, then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality
    Article Snippet: Paragraph title: RNA-Seq and Data Processing ... USER Enzyme (New England Biolabs) was subsequently used with size-selected, adaptor-ligated cDNA.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: Paragraph title: RNA sequencing ... To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Magnetic Beads:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Briefly, poly(A)+ mRNA was isolated from 500 ng of total RNA on oligo(dT) magnetic beads. .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Briefly, purified mRNA was obtained from total RNA (1 µg) per sample using poly-T oligo-attached magnetic beads; then, the mRNA was fragmented using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality
    Article Snippet: Briefly, mRNA was enriched using oligo (dT)-attached magnetic beads. .. USER Enzyme (New England Biolabs) was subsequently used with size-selected, adaptor-ligated cDNA.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: After purified with poly-T oligo-attached magnetic beads, the mRNAs were fragmented. .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: The mRNA was purified using poly-T oligo-attached magnetic beads followed by fragmentation using NEBNext First Strand Synthesis Reaction Buffer (5x). .. To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Isolation:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Paragraph title: Temperature Shift, RNA Isolation, Primer Extension, and RNA Sequence (RNA-Seq) Analyses ... Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: Paragraph title: RNA isolation, library construction and sequencing ... Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: Total RNA isolation was performed with TRIzol reagent (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. .. Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C.

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB). .. Proteins were expressed, and microsomal fractions containing the recombinant protein were isolated as described previously ( ; ; ).

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: Paragraph title: RNA isolation, cDNA library construction, and sequencing ... Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    Purification:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Double-stranded cDNA was purified with AMPure XP beads and applied to the standard Illumina library protocol including end repair, dA tailing, and ligation with Illumina sequencing adapters. .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C. .. Then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: In order to select cDNA fragments of preferentially 150~ 200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, US). .. Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Integrative Analyses of Long Non-coding RNA and mRNA Involved in Piglet Ileum Immune Response to Clostridium perfringens Type C Infection
    Article Snippet: To select cDNA fragments of preferentially 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR, then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: In order to select cDNA fragments of preferentially 150~ 200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality
    Article Snippet: In order to select cDNA fragments of the appropriate size, library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, MA, United States). .. USER Enzyme (New England Biolabs) was subsequently used with size-selected, adaptor-ligated cDNA.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: Library fragments were then purified using AMPure XP system (Beckman Coulter, Beverly, MA, United States), in order to preferentially select cDNA fragments of 150–200 bp in length. .. Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C.

    Article Title: Full Mitogenomes in the Critically Endangered Kākāpō Reveal Major Post-Glacial and Anthropogenic Effects on Neutral Genetic Diversity
    Article Snippet: For historical specimens, double stranded Illumina libraries were prepared according to Meyer & Kircher [ ] using uracil-treatment with the USER enzyme (New England Biolabs, Ipswich, MA, USA) and indexing PCRs with AccuPrimeTM Pfx DNA Polymerase (Life Technologies, Carlsbad, CA, USA). .. Purification and size selection of libraries was performed using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), and their concentrations were measured with a high-sensitivity DNA chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: To select cDNA fragments of 250–350 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter). .. Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: To preferentially select cDNA fragments of 150–200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: To preferentially select insert fragments that were 150~200 bp in length, the library fragments were purified with AMPure XP beads (Beckman Coulter, Beverly, USA). .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: The library fragments were purified, for a 150–200 bp size range, with AMpure XP purification kit (Beckman Coulter, MA, USA). .. To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Polymerase Chain Reaction:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs). .. Following PCR enrichment, libraries underwent fragment size confirmation using an HS DNA chip on an Agilent BioAnalyzer and were quantified using the Illumina library quantification kit (KAPA).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C. .. Then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: .. Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Integrative Analyses of Long Non-coding RNA and mRNA Involved in Piglet Ileum Immune Response to Clostridium perfringens Type C Infection
    Article Snippet: .. Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR, then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. .. At last, products were purified (AMPure XP system) and library quality were assessed on the Agilent Bioanalyzer 2100 system.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Article Title: Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality
    Article Snippet: USER Enzyme (New England Biolabs) was subsequently used with size-selected, adaptor-ligated cDNA. .. Then, PCR was carried out with Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer.

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C. .. PCR was then performed using Phusion High-Fidelity DNA polymerase with Universal PCR primers and Index (X) Primer.

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C. .. PCR was then performed using the Phusion High-Fidelity DNA polymerase (Thermo Scientific), Universal PCR primers (New England BioLabs), and the Index (X) Primer (New England BioLabs).

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: .. PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest. .. In order to utilize the URA3 + pGO45 and pMM134 plasmids, which express GFP-CPS or Sna3p-GFP, respectively [ , ], for yeast endosome:vacuole transport studies in the presence of another URA3 + yeast expression plasmid harboring the putative w Bm effector, pGO45 and pMM134 were first converted from URA3 + to LYS2 + via homologous recombination methods by digesting pM2660 (ATCC) with HindIII [ ], and transforming yeast strains harboring either pGO45 or pMM134 with this digest.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR. .. Then, PCR was performed with a Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) primer.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR. .. PCR was then performed with Phusion high-fidelity DNA polymerase, universal PCR primers and the index (X) primer.

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system
    Article Snippet: .. The GYS PCR primers contained USER enzyme (NEB) cleavage sites. .. RT-PCR products were cleaved with USER enzyme (NEB), mixed with pNEB206A USER vector and transformed into competent E. coli strain NEB5alpha (NEB).

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C. .. The cDNA was enriched by PCR reactions using Phusion High-Fidelity DNA polymerase, universal PCR primers and index primers.

    cDNA Library Assay:

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: Paragraph title: RNA isolation, cDNA library construction, and sequencing ... Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: Paragraph title: cDNA library construction and Illumina sequencing ... Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Chromatin Immunoprecipitation:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs). .. Following PCR enrichment, libraries underwent fragment size confirmation using an HS DNA chip on an Agilent BioAnalyzer and were quantified using the Illumina library quantification kit (KAPA).

    Article Title: Full Mitogenomes in the Critically Endangered Kākāpō Reveal Major Post-Glacial and Anthropogenic Effects on Neutral Genetic Diversity
    Article Snippet: For historical specimens, double stranded Illumina libraries were prepared according to Meyer & Kircher [ ] using uracil-treatment with the USER enzyme (New England Biolabs, Ipswich, MA, USA) and indexing PCRs with AccuPrimeTM Pfx DNA Polymerase (Life Technologies, Carlsbad, CA, USA). .. Purification and size selection of libraries was performed using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), and their concentrations were measured with a high-sensitivity DNA chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).

    Plasmid Preparation:

    Article Title: The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1The Gymnosperm Cytochrome P450 CYP750B1 Catalyzes Stereospecific Monoterpene Hydroxylation of (+)-Sabinene in Thujone Biosynthesis in Western Redcedar 1 [OPEN]
    Article Snippet: A cassette for Uracil-Specific Excision Reagent (USER)-based cloning was inserted into multicloning site1 (MSC1) of the pESC-LEU2d vector ( ) using the Bam HI and Hin dIII site based on . .. CYP76AA25 and CYP750B1 s were cloned into Pac I, and Nb.BbvC ) and treatment with USER enzyme (NEB).

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: .. PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest. .. In order to utilize the URA3 + pGO45 and pMM134 plasmids, which express GFP-CPS or Sna3p-GFP, respectively [ , ], for yeast endosome:vacuole transport studies in the presence of another URA3 + yeast expression plasmid harboring the putative w Bm effector, pGO45 and pMM134 were first converted from URA3 + to LYS2 + via homologous recombination methods by digesting pM2660 (ATCC) with HindIII [ ], and transforming yeast strains harboring either pGO45 or pMM134 with this digest.

    Article Title: Controlled expression of functional miR-122 with a ligand inducible expression system
    Article Snippet: The GYS PCR primers contained USER enzyme (NEB) cleavage sites. .. RT-PCR products were cleaved with USER enzyme (NEB), mixed with pNEB206A USER vector and transformed into competent E. coli strain NEB5alpha (NEB).

    RNA Extraction:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: Paragraph title: 2.2. RNA Extraction, Library Preparation, Sequencing and De Novo Assembly ... The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Selection:

    Article Title: Full Mitogenomes in the Critically Endangered Kākāpō Reveal Major Post-Glacial and Anthropogenic Effects on Neutral Genetic Diversity
    Article Snippet: For historical specimens, double stranded Illumina libraries were prepared according to Meyer & Kircher [ ] using uracil-treatment with the USER enzyme (New England Biolabs, Ipswich, MA, USA) and indexing PCRs with AccuPrimeTM Pfx DNA Polymerase (Life Technologies, Carlsbad, CA, USA). .. Purification and size selection of libraries was performed using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), and their concentrations were measured with a high-sensitivity DNA chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).

    Sample Prep:

    Article Title: Global Transcriptomic Analysis and Function Identification of Malolactic Enzyme Pathway of Lactobacillus paracasei L9 in Response to Bile Stress
    Article Snippet: For RNA sample preparation, 3 μg RNA per sample was used as input material. .. Size-selected and adaptor-ligated cDNA was then treated with 3 μl USER Enzyme (NEB, United States) at 37°C for 15 min followed by 5 min at 95°C.

    Ethanol Precipitation:

    Article Title: Integrative Analyses of Long Non-coding RNA and mRNA Involved in Piglet Ileum Immune Response to Clostridium perfringens Type C Infection
    Article Snippet: Ribosomal RNA (rRNA) was removed from total RNA by epicenter Ribo-zero™ rRNA Removal Kit (epicenter, USA), and rRNA free residue was cleaned up by ethanol precipitation. .. Then 3 μL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR, then PCR reaction was performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer.

    Random Hexamer Labeling:

    Article Title: Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription *
    Article Snippet: Purified RNA was fragmented, annealed with random hexamer primers, and applied as template for first strand cDNA synthesis by SuperScript II reverse transcriptase (Invitrogen). .. Prior to PCR, dsDNA libraries were treated with USER enzyme to introduce single strand breaks (New England Biolabs).

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: First, strand cDNA was produced based on the random hexamer primer and M-MuLV Reverse Transcriptase. .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Article Title: Transcriptomic and metabolic flux analyses reveal shift of metabolic patterns during rice grain development
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). .. Then 3 μl USER Enzyme (NEB, US) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Genome-wide expression profiling of leaves and roots of watermelon in response to low nitrogen
    Article Snippet: First strand cDNA was synthesized using random hexamer primer and M-MuLV reverse transcriptase. .. Then 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Metabolomic and Transcriptomic Analyses Reveal That a MADS-Box Transcription Factor TDR4 Regulates Tomato Fruit Quality
    Article Snippet: These fragments were used to synthesize first-strand cDNA using random hexamer primers and M-MuLV Reverse Transcriptase. .. USER Enzyme (New England Biolabs) was subsequently used with size-selected, adaptor-ligated cDNA.

    Article Title: Transcriptomics reveals the molecular processes of light-induced rapid darkening of the non-obligate cave dweller Oreolalax rhodostigmatus (Megophryidae, Anura) and their genetic basis of pigmentation strategy
    Article Snippet: First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H−). .. Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C before PCR.

    Article Title: Screening and characterization of long noncoding RNAs involved in the albinism of Ananas comosus var. bracteatus leaves
    Article Snippet: First-strand cDNA was synthesized using a random hexamer primer and reverse transcriptase. .. Then, 3 μl of USER enzyme (NEB, USA) was incubated with the size-selected, adaptor-ligated cDNA at 37°C for 15 min before PCR.

    Article Title: An influential meal: host plant dependent transcriptional variation in the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae)
    Article Snippet: Synthesis of first strand cDNA was performed using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H− ), subsequently second strand cDNA was synthesized using DNA polymerase I and RNase H. After terminal repair of the sequences, the 3′ ends were adenylated and NEBnext adapters (NEB, MA, USA) were ligated. .. To the size-selected cDNA, 3 μl USER Enzyme (NEB, MA, USA) was added and heated to 37 °C for 15 min followed by 5 min at 95 °C.

    Spectrophotometry:

    Article Title: Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish †
    Article Snippet: The quantity and quality of total RNA were analyzed using a spectrophotometer (Nanodrop) and by gel electrophoresis. .. Next, the size-selected, adaptor-ligated cDNA was incubated with 3 μL of USER Enzyme (NEB) at 37°C for 15 min, followed by 5 min at 95°C.

    Produced:

    Article Title: De Novo Transcriptome Assembly and Population Genetic Analyses for an Endangered Chinese Endemic Acer miaotaiense (Aceraceae)
    Article Snippet: First, strand cDNA was produced based on the random hexamer primer and M-MuLV Reverse Transcriptase. .. The fragments in library were purified with AMPure XP system (Beckman Coulter, Danvers, MA, USA) to firstly select for 240 bp cDNA fragments; 3 μL USER Enzyme (New England Biolabs) was incubated with size-selected, adaptor-ligated cDNA for 15 min at 37 °C and then 5 min at 95 °C.

    Homologous Recombination:

    Article Title: Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi
    Article Snippet: PCR products were then treated with the USER Enzyme (New England Biolabs) to create unique 3´ single-stranded extensions which anneal and ligate to linearized pYES2/NT A, which had been previously amplified with vector-specific primers containing a complementary linker to the insert gene of interest. .. In order to utilize the URA3 + pGO45 and pMM134 plasmids, which express GFP-CPS or Sna3p-GFP, respectively [ , ], for yeast endosome:vacuole transport studies in the presence of another URA3 + yeast expression plasmid harboring the putative w Bm effector, pGO45 and pMM134 were first converted from URA3 + to LYS2 + via homologous recombination methods by digesting pM2660 (ATCC) with HindIII [ ], and transforming yeast strains harboring either pGO45 or pMM134 with this digest.

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    New England Biolabs wild type cas9 expression plasmid
    Genomic DNA modification by <t>intein-Cas9(S219),</t> intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are
    Wild Type Cas9 Expression Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Genomic DNA modification by intein-Cas9(S219), intein-Cas9(C574), and wild-type Cas9. ( a ) Indel frequency from high-throughput DNA sequencing of amplified genomic on-target sites in the absence or presence of 4-HT. Note that a significant number of indels were observed at the CLTA on-target site even in the absence of a targeting sgRNA ( Supplementary Table 7 ). ( b–d ) DNA modification specificity, defined as on-target:off-target indel frequency ratio 4 – 6 , normalized to wild-type Cas9. Cells were transfected with 500 ng of the Cas9 expression plasmid. P -values are

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Modification, High Throughput Screening Assay, DNA Sequencing, Amplification, Transfection, Expressing, Plasmid Preparation

    Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Journal: Nature chemical biology

    Article Title: Small Molecule-Triggered Cas9 Protein with Improved Genome-Editing Specificity

    doi: 10.1038/nchembio.1793

    Figure Lengend Snippet: Insertion of an evolved ligand-dependent intein enables small-molecule control of Cas9. ( a ) Intein insertion renders Cas9 inactive. Upon 4-HT binding, the intein undergoes conformational changes that trigger protein splicing and restore Cas9 activity. ( b ) The evolved intein was inserted to replace each of the colored residues. Intein-inserted Cas9 variants at S219 and C574 (green) were used in subsequent experiments. ( c ) Genomic EGFP disruption activity of wild-type Cas9 and intein-Cas9 variants in the absence or presence of 4-HT. Intein-Cas9 variants are identified by the residue replaced by the intein. Error bars reflect the standard deviation of three biological replicates.

    Article Snippet: Intein 37R3-2 was subcloned at the described positions into the wild-type Cas9 expression plasmid using USER (NEB M5505) cloning. sgRNA expression plasmids used in this study have been described previously .

    Techniques: Binding Assay, Activity Assay, Standard Deviation

    Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Journal: Nucleic Acids Research

    Article Title: A versatile element for gene addition in bacterial chromosomes

    doi: 10.1093/nar/gkr1085

    Figure Lengend Snippet: Design of primers for USER cloning with pMS26. Two choices of translation signal in the 5′-UTR are shown. The gene-specific sequence illustrated is of fnuDIIM . The underlined 21 bp of longer translation signal (LTS) is from the tacp regulatory region ( 34 ) ; the short signal (STS) is a truncation of it. The LTS and downstream primers illustrated are the same as primers 5 and 6 of Table 4 ; the STS construct was made but not used in this report. Fusion of lacZ to the signal as shown creates an RBS/ATG spacing of six, within the usual range of spacing ( 35 ); lacZ native spacing is seven ( 36 ).

    Article Snippet: General materials: ∘ USERBstBI-compatible digested pMS26 (from step 1) ∘ PfuCx_TurboCx _Hotstart_DNA_polymerase (Agilent Genomics) ∘ USER enzyme (NEB M5505) ∘ PCR purification columns ∘ Universal flanking primers (glmS, ptsS ) to monitor chromosomal insertion ∘ RB ampicillin plates ∘ RB no drug plates ∘ Incubators at 30°C and 42°C ∘ SOC or other outgrowth medium Experiment-specific materials: ∘ competent host cells ∘ DNA template ∘ gene-specific primers with 5′ sequences suitable to generate USERBstBI-compatible extensions.

    Techniques: Clone Assay, Sequencing, Construct

    Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Journal: PLoS ONE

    Article Title: Inhibitors of MyD88-Dependent Proinflammatory Cytokine Production Identified Utilizing a Novel RNA Interference Screening Approach

    doi: 10.1371/journal.pone.0007029

    Figure Lengend Snippet: Schematic diagram of random shRNA library construction. (A) Backbone of oligonucleotide used for generation of shRNA library. (B.1–2) First, the 120 bp oligonucleotide containing 20 bp of the 3′ end of U6 including a “G” to initiate transcription, 18 random nucleotides (sense) and a stem-loop structure that can act as a primer for synthesizing the strand complementary to the random 18 bp (anti-sense) was extended using T4 DNA polymerase in the presence of a blocking primer which annealed to the U6 promoter region. (B.3–4) Following purification of the extended oligonucleotide, a poly-thymidine tract was added using terminal transferase (TdT). (B.5) Exo - klenow fragment was used to make the oligonucleotide double stranded using a poly-A oligonucleotide as a primer. (B.6) The purified double stranded DNA was amplified using uracil containing primers. (B.7) The PCR product was digested with USER enzyme to generate overhangs to facilitate cloning. (B.8) The PCR fragment was cloned into the lentiviral vector pLL3.7, and digested with BpmI to remove the extra sequence between the random sense and antisense sequence, leaving a 9 base pair loop sequence.

    Article Snippet: Step 7 To generate cohesive ends, the amplified PCR product was treated with the USER enzyme (New England Biolabs) according to the manufacturer's instructions, which specifically removes deoxyuridine in the DNA.

    Techniques: shRNA, Activated Clotting Time Assay, Blocking Assay, Purification, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Sequencing

    Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications

    doi: 10.1093/dnares/dsw038

    Figure Lengend Snippet: Amplification of uracils with different Phusion polymerases with smPCR. The amplification efficiency of Phusion Hot Start II and Phusion U was compared for samples that contain uracil in both strands (forward and reverse; HSI_insert_1 construct). Efficiency was measured as the percentage of positive smPCR reactions. In total, 372 smPCR reactions were analyzed for each condition (without USER treatment, and USER treatment before amplification). Error bars represent Poisson 95% CIs.

    Article Snippet: Treatments with the USER enzyme were performed on HSI_insert_1 by incubating 2 × 107 copies HSI_insert construct with 1 U USER enzyme (NEB) in 1× Phusion HF Buffer in a reaction volume of 20 µl at 37 °C for 30 min prior to amplification.

    Techniques: Amplification, Construct