superhelical puc19  (New England Biolabs)


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    Name:
    pUC19 Vector
    Description:
    pUC19 Vector 250 ug
    Catalog Number:
    n3041l
    Price:
    294
    Size:
    250 ug
    Category:
    Vectors Plasmids
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    Structured Review

    New England Biolabs superhelical puc19
    pUC19 Vector
    pUC19 Vector 250 ug
    https://www.bioz.com/result/superhelical puc19/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    superhelical puc19 - by Bioz Stars, 2020-01
    90/100 stars

    Related Products / Commonly Used Together

    na-l-ascorbate
    nacl
    hepes buffer

    Images

    1) Product Images from "DNA oxidation profiles of copper phenanthrene chemical nucleases"

    Article Title: DNA oxidation profiles of copper phenanthrene chemical nucleases

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2015.00028

    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.
    Figure Legend Snippet: Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Techniques Used: Incubation, Binding Assay

    Related Articles

    Clone Assay:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Cloned plasmids were delivered to the E5 mutant by electroporation.

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed. .. The clones were used in separate in vitro transcription reactions, after which the individual ssRNAs were combined, annealed to create dsRNA, and purified according to the manufacturer’s protocol.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs). .. The dCas9-Tet1 plasmid was constructed based on the parental plasmid PX458.

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: Paragraph title: Cloning, mutagenesis and in vitro transcription ... For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. The sequence of the recombinant plasmid was determined to ensure that no errors had been introduced.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19. .. 1LTR TOPO vector was digested with EcoRI and NarI, and the fragment with LTR sequence was introduced into the region between EcoRI and SmaI of 1LTR puc19 vector to create a 2LTR puc19 vector.

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
    Article Snippet: .. BPV1 nt 7914 to 27 were cloned into Xba I and Hind III restriction enzyme sites of the pUC19 vector (New England Biolabs) to generate pBPV1ori (so-called because the LCR fragment cloned contains the minimal E1-dependent origin of replication). .. The Avr II-to- Bam HI DNA fragment from plasmid pBS1033 containing the basic BamC promoter from EBV fused to the firefly luciferase gene was cloned into the Xba I and Bam HI restriction enzyme sites of pBPV1ori to generate pKT260.

    Amplification:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: .. The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Another fragment of the b2TUB promoter-Aph7 gene-RbcS2 terminator, which confers resistance to hygromycin, was amplified with the primers Aph_Gibson_fwd and Aph_Gibson_rev and then assembled into the pUC19 vector backbone harboring the CpSRP43 cassette, which was amplified with the primers Aph-pUC_back_fwd and Aph-pUC_back_rev.

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: The DvSnf7 was amplified from a sequence-confirmed plasmid using a high fidelity polymerase and the single product was gel extracted using a Qiagen Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA). .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Homology arms were PCR amplified from genomic DNA extracted from NIH3T3 (ATCC, USA) cells and cloned into EcoRI (R0101S, New England Biolabs) and SalI (R0138S, New England Biolabs) digested pUC19 vector (EZ002S, Enzynomics). .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: An annealing temperature of 57°C amplified a 2,169-bp genomic region which contained the putative sct gene and the putative d -citramalyl-CoA lyase ( ccl ) gene. .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs).

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: Amplification was performed in a T100 Thermo Cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using a 30 s initial denaturation at 98 °C, 30 cycles of 10 s denaturation at 98 °C, 15 s annealing at 60 °C, 30 s extension at 72 °C, and a final 2 min extension at 72 °C. .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: Coding regions of human MAP4K4 and SRSF3 were amplified using a polymerase chain reaction (PCR) with a mice fetal complementary (c)DNA library as the template. .. The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB).

    Positive Control:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    Synthesized:

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed. .. In vitro synthesized dsRNA was quantified by Nanodrop (Thermo Scientific, Wilmington, DE) at 260 nm and purity was evaluated by examining the 260/280 nm ratios.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs). .. The dCas9-Tet1 plasmid was constructed based on the parental plasmid PX458.

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: Cloning, mutagenesis and in vitro transcription A bicistronic DNA template ( ) encoding the well-characterized B. subtilis glyQS T-box leader (+10–182) ( , , , ), as well as its cognate tRNAGly ( , , , ), was synthesized by GenScript (Piscataway, NJ, USA). .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

    Lambda DNA Preparation:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Generation of spike-in controls for 4mC-TAB-seq and MethylC-seq of C. kristjanssonii C/5mC spike-in control (CpG methylated lambda DNA) was prepared by treating unmethylated lambda cl857 DNA (Promega) with M. SssI (NEB) according to the manufacturer's instructions. .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG).

    TA Cloning:

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: This product was then TA cloned into the pCR4-TOPO vector using a TA cloning kit (Thermo Fisher Scientific), resulting in 1LTR TOPO, and sequenced. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Construct:

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Donor constructs for genome editing were generated consisting of upper and lower arms (1600 bp) homologous to the targeted cut site. .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: In addition, another variant of this bicistronic construct was generated in which the less conserved Stem III helix was elongated by adding 10 bp just before the loop spanning nucleotides 134–139 (Figure ) (10 bases before 134 and 10 complementary bases after 139) (S3.10), by using standard site-directed mutagenesis protocols ( ). .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: PCR amplification of the gene construct was carried out with Q5 High-Fidelity Master Mix (New England Biolabs Inc., Ipswich, MA, USA) using the forward tag primer (5′-GCTTGCATCGTACGTATCGG-3′), and the reverse tag primer (5′-AGACGTAACGACCAACGCTAG-3′). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Article Title: Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin
    Article Snippet: .. The deleting construct based on pUC19 vector was delivered into diploid S . cerevisiae MH272a/α strain, and transformants were selected using minus-leucine minimal medium [ ]. .. Disruptions were confirmed with the primers annealing outside recombination area by PCR with chromosomal DNA isolated from the engineered yeast.

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: A restriction enzyme map of M2-67-B11 fosmid was constructed by digestion of middle and end of each putative open reading frame (ORF) with restriction enzymes, SbfI, SacI, PstI, and AvaI. .. To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan).

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: .. The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. PCR-amplified fragments were digested with EcoR I and Hind III restriction enzymes prior to DNA ligation.

    Incubation:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. Cells were re-suspended in 500 μl of super optimal broth with catabolite repressor (SOC) medium (20 g tryptone, 0.5 g yeast extract, 0.5 g 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2 , and 10 mM MgSO4 in 1 L of deionized water adjusted to a final pH 7.0) and incubated for 1 h at 37 °C before plating.

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: Two PCR products were then mixed at a 1:1 ratio and incubated at 95°C for 5 minutes before being cooled down to the room temperature to allow annealing of the 2 products at the shared R region. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB). .. Mixtures of third strands (10 μM) and plasmid (10 nM) in the standard buffer were incubated at 37°C for 2 h and then overnight at 4°C.

    Luciferase:

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
    Article Snippet: The Avr II-to- Bam HI DNA fragment from plasmid pBS1033, also referred to as oriP-BamH1 C-Luc' , containing the “basic” BamC promoter from Epstein Barr virus (EBV) fused to the firefly luciferase gene, was cloned into the Spe I and Bam HI restriction enzyme sites of pKT266 to generate pKT267. .. BPV1 nt 7914 to 27 were cloned into Xba I and Hind III restriction enzyme sites of the pUC19 vector (New England Biolabs) to generate pBPV1ori (so-called because the LCR fragment cloned contains the minimal E1-dependent origin of replication).

    Activity Assay:

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs). .. Two nuclease activity sites of Cas9 were mutated by the site-directed mutagenesis method (D10A, H840A).

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan). .. The plasmid containing ORF related toxoflavin-degrading activity was named as pUC-TxeA.

    Expressing:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: .. The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Another fragment of the b2TUB promoter-Aph7 gene-RbcS2 terminator, which confers resistance to hygromycin, was amplified with the primers Aph_Gibson_fwd and Aph_Gibson_rev and then assembled into the pUC19 vector backbone harboring the CpSRP43 cassette, which was amplified with the primers Aph-pUC_back_fwd and Aph-pUC_back_rev.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Plasmids construction For targeting mouse Oct4 and inducing the DSB on the target region, the single guide RNA (sgRNA) coding sequence was incorporated into the BbsI (R0539S, New England Biolabs) site of the bicistronic expression plasmids PX458 or PX459, respectively (#48138, #48139, Addgene). .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: Paragraph title: Cloning and expression of a putative succinyl-CoA: d -citramalate CoA transferase ( sct ) gene in E. coli BL21. ... The PCR product was purified and cloned into pUC19 vector (New England Biolabs).

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. Derived mutants of the expressing vectors or minigene reporters harboring substituted nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands).

    Modification:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Transformation Assay:

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites. .. The resulting plasmid was transformed into DH5α cells and purified by standard protocols ( ).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. Competent E. coli BL21 cells ( ) were transformed with pAST2, grown at 37°C in Luria-Bertani medium containing 100 μg of ampicillin ml−1 , and induced at an optical density of 0.8 with 0.4 mM isopropyl-β- d -thiogalactopyranoside.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    Derivative Assay:

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
    Article Snippet: BPV1 nt 7914 to 27 were cloned into Xba I and Hind III restriction enzyme sites of the pUC19 vector (New England Biolabs) to generate pBPV1ori (so-called because the LCR fragment cloned contains the minimal E1-dependent origin of replication). .. The plasmid p1073 is closely derived from pBS1013 and has four consensus BPV1 E2 binding sites positioned upstream of the tk promoter (T. Gahn, unpublished data).

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. Derived mutants of the expressing vectors or minigene reporters harboring substituted nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands).

    Electroporation:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Cloned plasmids were delivered to the E5 mutant by electroporation.

    DNA Ligation:

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. PCR-amplified fragments were digested with EcoR I and Hind III restriction enzymes prior to DNA ligation.

    Cell Culture:

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan). .. Each transformant was cultured in LB medium containing toxoflavin of 30 μg/mL and measured the optical density at 600 nm.

    Generated:

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Donor constructs for genome editing were generated consisting of upper and lower arms (1600 bp) homologous to the targeted cut site. .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: In addition, another variant of this bicistronic construct was generated in which the less conserved Stem III helix was elongated by adding 10 bp just before the loop spanning nucleotides 134–139 (Figure ) (10 bases before 134 and 10 complementary bases after 139) (S3.10), by using standard site-directed mutagenesis protocols ( ). .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

    Polymerase Chain Reaction:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. All fragments amplified by PCR were used after purification with a QIAquick gel extraction kit (Qiagen, Netherlands).

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: This PCR product was used in the in vitro transcription reaction to create dsRNA which was purified according to manufacturer’s instructions. .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Homology arms were PCR amplified from genomic DNA extracted from NIH3T3 (ATCC, USA) cells and cloned into EcoRI (R0101S, New England Biolabs) and SalI (R0138S, New England Biolabs) digested pUC19 vector (EZ002S, Enzynomics). .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. The sequence of the recombinant plasmid was determined to ensure that no errors had been introduced.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: A complete LTR with partial env sequence on the 5′ side and primer binding site on the 3′ side was obtained by overlapping PCR of above product by Platinum Taq Hi Fidelity Kit using Bgl-F and Gag-R as primers. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The PCR product was digested with EcoR V and Sac I restriction enzymes, and then the insert was placed into the pUC19 vector (NEB). .. The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB).

    Sequencing:

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed. .. The clones were used in separate in vitro transcription reactions, after which the individual ssRNAs were combined, annealed to create dsRNA, and purified according to the manufacturer’s protocol.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Plasmids construction For targeting mouse Oct4 and inducing the DSB on the target region, the single guide RNA (sgRNA) coding sequence was incorporated into the BbsI (R0539S, New England Biolabs) site of the bicistronic expression plasmids PX458 or PX459, respectively (#48138, #48139, Addgene). .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: On the basis of a protein sequence alignment of the BbsF ( R )-benzylsuccinate CoA-transferase (GenBank accession number ) from Thauera aromatica , a highly conserved region on the C. aurantiacus genome was found located next to the gene of a putative 3-hydroxy-3-methylglutaryl-CoA lyase. .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs).

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: A complete LTR with partial env sequence on the 5′ side and primer binding site on the 3′ side was obtained by overlapping PCR of above product by Platinum Taq Hi Fidelity Kit using Bgl-F and Gag-R as primers. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: Based on the sequencing results, a toxoflavin-degrading fosmid was selected and named as M2-67-B11. .. To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan).

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Binding Assay:

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: The corresponding binding sites of the four sgRNA and their locations are described in Supple. .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs).

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: A complete LTR with partial env sequence on the 5′ side and primer binding site on the 3′ side was obtained by overlapping PCR of above product by Platinum Taq Hi Fidelity Kit using Bgl-F and Gag-R as primers. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: Paragraph title: Third strand binding to plasmid DNA ... A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB).

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
    Article Snippet: BPV1 nt 7914 to 27 were cloned into Xba I and Hind III restriction enzyme sites of the pUC19 vector (New England Biolabs) to generate pBPV1ori (so-called because the LCR fragment cloned contains the minimal E1-dependent origin of replication). .. The plasmid p1073 is closely derived from pBS1013 and has four consensus BPV1 E2 binding sites positioned upstream of the tk promoter (T. Gahn, unpublished data).

    Transmission Electron Microscopy:

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Nucleic Acid Electrophoresis:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: PCR products were column purified using the GenCatch PCR Cleanup Kit (Epoch Life Science., Sugar Land, TX, USA) and verified using gel electrophoresis in a 1% (w/v) agarose gel in 1x TAE buffer run at 100 V for 30 min. .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Methylation:

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: Generation of spike-in controls for 4mC-TAB-seq and MethylC-seq of C. kristjanssonii C/5mC spike-in control (CpG methylated lambda DNA) was prepared by treating unmethylated lambda cl857 DNA (Promega) with M. SssI (NEB) according to the manufacturer's instructions. .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG).

    Mutagenesis:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Cloned plasmids were delivered to the E5 mutant by electroporation.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs). .. Two nuclease activity sites of Cas9 were mutated by the site-directed mutagenesis method (D10A, H840A).

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: Paragraph title: Cloning, mutagenesis and in vitro transcription ... For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: .. A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB). .. After purifying with a Qiagen plasmid purification kit, the plasmid was shown by agarose gel electrophoresis to be at least 70% supercoiled.

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. Derived mutants of the expressing vectors or minigene reporters harboring substituted nucleotides were constructed using the QuikChange site-directed mutagenesis system (Stratagene, Amsterdam, The Netherlands).

    Isolation:

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: Plasmid DNA was isolated by the method of Birnboim and Doly ( ). .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs).

    Purification:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. All fragments amplified by PCR were used after purification with a QIAquick gel extraction kit (Qiagen, Netherlands).

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: This PCR product was used in the in vitro transcription reaction to create dsRNA which was purified according to manufacturer’s instructions. .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed.

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites. .. The resulting plasmid was transformed into DH5α cells and purified by standard protocols ( ).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. The sequence of the recombinant plasmid was determined to ensure that no errors had been introduced.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: PCR products were column purified using the GenCatch PCR Cleanup Kit (Epoch Life Science., Sugar Land, TX, USA) and verified using gel electrophoresis in a 1% (w/v) agarose gel in 1x TAE buffer run at 100 V for 30 min. .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB). .. After purifying with a Qiagen plasmid purification kit, the plasmid was shown by agarose gel electrophoresis to be at least 70% supercoiled.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: The viral RNA was then reverse transcribed by ThermoScript RT-PCR System (Thermo Fisher Scientific) using Gag-R (all primer sequences are listed in ) or R-R reverse primers, covering 5′ and 3′ LTR sequences. .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19.

    Recombinant:

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. The sequence of the recombinant plasmid was determined to ensure that no errors had been introduced.

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: .. To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan). .. Each transformant was cultured in LB medium containing toxoflavin of 30 μg/mL and measured the optical density at 600 nm.

    Staining:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: The resulting gel was stained with GelRed (Thermo Fisher Scientific Inc., Waltham, MA, USA). .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Mouse Assay:

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: Coding regions of human MAP4K4 and SRSF3 were amplified using a polymerase chain reaction (PCR) with a mice fetal complementary (c)DNA library as the template. .. The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB).

    Plasmid Preparation:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: .. The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. Another fragment of the b2TUB promoter-Aph7 gene-RbcS2 terminator, which confers resistance to hygromycin, was amplified with the primers Aph_Gibson_fwd and Aph_Gibson_rev and then assembled into the pUC19 vector backbone harboring the CpSRP43 cassette, which was amplified with the primers Aph-pUC_back_fwd and Aph-pUC_back_rev.

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed. .. The clones were used in separate in vitro transcription reactions, after which the individual ssRNAs were combined, annealed to create dsRNA, and purified according to the manufacturer’s protocol.

    Article Title: Regulation of gene expression by altered promoter methylation using a CRISPR/Cas9-mediated epigenetic editing system
    Article Snippet: .. Then, synthesized a 260-mer oligonucleotide containing altered CpG dinucleotides and reporter sites was cloned into the above donor pUC19 vector following digestion with AfeI (R0652L, New England Biolabs) and BamHI (R0136S, New England Biolabs). .. The dCas9-Tet1 plasmid was constructed based on the parental plasmid PX458.

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites. .. The resulting plasmid was transformed into DH5α cells and purified by standard protocols ( ).

    Article Title: Properties of Succinyl-Coenzyme A:d-Citramalate Coenzyme A Transferase and Its Role in the Autotrophic 3-Hydroxypropionate Cycle of Chloroflexus aurantiacus
    Article Snippet: .. The PCR product was purified and cloned into pUC19 vector (New England Biolabs). .. The sequence of the recombinant plasmid was determined to ensure that no errors had been introduced.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs). .. The digested products were then cleansed of extraneous DNA using the MinElute Reaction Cleanup Kit (QIAGEN, Germantown, MD).

    Article Title: Enhanced antagonism of BST-2 by a neurovirulent SIV envelope
    Article Snippet: .. To create 2LTR puc19 with unique restriction sites that allow the rest of the coding regions to be transferred, 1LTR TOPO clones were digested with NdeI and NarI restriction enzymes and transferred into puc19 vector (New England Biolabs), resulting in 1LTR puc19. .. 1LTR TOPO vector was digested with EcoRI and NarI, and the fragment with LTR sequence was introduced into the region between EcoRI and SmaI of 1LTR puc19 vector to create a 2LTR puc19 vector.

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: .. A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB). .. After purifying with a Qiagen plasmid purification kit, the plasmid was shown by agarose gel electrophoresis to be at least 70% supercoiled.

    Article Title: Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin
    Article Snippet: .. The deleting construct based on pUC19 vector was delivered into diploid S . cerevisiae MH272a/α strain, and transformants were selected using minus-leucine minimal medium [ ]. .. Disruptions were confirmed with the primers annealing outside recombination area by PCR with chromosomal DNA isolated from the engineered yeast.

    Article Title: Isolation and characterization of a novel metagenomic enzyme capable of degrading bacterial phytotoxin toxoflavin
    Article Snippet: .. To find the ORF responsible for toxoflavin degradation, each DNA fragment was ligated into pUC19 vector (NEB, England) and then the recombinant plasmids were introduced into E .coli XL1-Blue cell (RBC, Taiwan). .. Each transformant was cultured in LB medium containing toxoflavin of 30 μg/mL and measured the optical density at 600 nm.

    Article Title: Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing
    Article Snippet: .. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). ..

    Article Title: Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
    Article Snippet: .. BPV1 nt 7914 to 27 were cloned into Xba I and Hind III restriction enzyme sites of the pUC19 vector (New England Biolabs) to generate pBPV1ori (so-called because the LCR fragment cloned contains the minimal E1-dependent origin of replication). .. The Avr II-to- Bam HI DNA fragment from plasmid pBS1033 containing the basic BamC promoter from EBV fused to the firefly luciferase gene was cloned into the Xba I and Bam HI restriction enzyme sites of pBPV1ori to generate pKT260.

    Article Title: RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution
    Article Snippet: .. The modified pUC19 vector was constructed by generating NcoI and NotI sites at the terminal ends of the pUC19 wild-type TEM-1 gene sequence by site directed mutagenesis (QuickChange Mutagenesis) to allow the wild-type TEM-1 gene to be deleted (leaving the original upstream regulating sequences intact). .. 10 ng of the cloned DNA was used to transform E. coli XL10-Gold cells (Stratagene) with an estimated transformation efficiency 5 × 109 colonies per ug pUC19 DNA.

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: .. The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. PCR-amplified fragments were digested with EcoR I and Hind III restriction enzymes prior to DNA ligation.

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: .. Two control experiments were also conducted: a positive control consisting of electrocompetent ΔcysE cells transformed with cysE in pUC19 vector and the other with electrocompetent ΔcysKΔcysM cells transformed with codon optimized cysM in pUC19 vector. .. The second positive control group supplemented the lack of cysteine through growing the knockout cells with empty pUC19 vectors on a fully supplemented media, LB + Amp, and were allowed to incubate at 37 °C overnight.

    shRNA:

    Article Title: RBM4a-SRSF3-MAP4K4 Splicing Cascade Constitutes a Molecular Mechanism for Regulating Brown Adipogenesis
    Article Snippet: The MAP4K4 minigene was constructed by inserting mouse MAP4K4 genomic fragments containing exon 15, exon16, exon 18, and the complete introns into the pUC19 vector (NEB). .. The vector-based short hairpin RNA targeting mouse SRSF3 was purchased from the RNAi core facility at Academia Sinica (Taipei, Taiwan).

    Agarose Gel Electrophoresis:

    Article Title: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes
    Article Snippet: PCR products were column purified using the GenCatch PCR Cleanup Kit (Epoch Life Science., Sugar Land, TX, USA) and verified using gel electrophoresis in a 1% (w/v) agarose gel in 1x TAE buffer run at 100 V for 30 min. .. For cloning and transformation, both the PCR products and the pUC19 vector (New England Biolabs) were digested using HindIII-HF and XhoI restriction enzymes at 37 °C for 1 h in 1x CutSmart buffer (New England Biolabs).

    Article Title: Repairing the Sickle Cell mutation. II. Effect of psoralen linker length on specificity of formation and yield of third strand-directed photoproducts with the mutant target sequence
    Article Snippet: A plasmid containing the Sickle Cell gene target with the mutant base pair was created by inserting a fragment consisting of the base pairs formed by residues 1–29 of strand D-2 (Fig. a) plus a 5′ AATT sticky end and its complement with a 3′ TCGA sticky end into a pUC19 vector between the HindIII and EcoRI sites using DNA ligase (NEB). .. After purifying with a Qiagen plasmid purification kit, the plasmid was shown by agarose gel electrophoresis to be at least 70% supercoiled.

    In Vitro:

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: This PCR product was used in the in vitro transcription reaction to create dsRNA which was purified according to manufacturer’s instructions. .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed.

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites. .. The resulting plasmid was transformed into DH5α cells and purified by standard protocols ( ).

    Gel Extraction:

    Article Title: Complementation of a mutation in CpSRP43 causing partial truncation of light-harvesting chlorophyll antenna in Chlorella vulgaris
    Article Snippet: The amplified fragments were assembled into the pUC19 vector amplified with the primers pUC19_fwd and pUC19_rev using a Gibson Assembly kit (NEB, USA) so that the gene expression was controlled by the 29B HSP70A promoter and Nos terminator. .. All fragments amplified by PCR were used after purification with a QIAquick gel extraction kit (Qiagen, Netherlands).

    Article Title: Characterization of the spectrum of insecticidal activity of a double-stranded RNA with targeted activity against Western Corn Rootworm (Diabrotica virgifera virgifera LeConte)
    Article Snippet: The DvSnf7 was amplified from a sequence-confirmed plasmid using a high fidelity polymerase and the single product was gel extracted using a Qiagen Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA). .. To create the clones, DNA fragments of the desired sequences with a single T7 polymerase promoter were cloned into the pUC19 vector (New England Biolabs, Ipswich, MA) between the EcoRI and HindIII restriction endonucleases recognition sites and were sequence confirmed.

    Variant Assay:

    Article Title: Molecular envelope and atomic model of an anti-terminated glyQS T-box regulator in complex with tRNAGly
    Article Snippet: In addition, another variant of this bicistronic construct was generated in which the less conserved Stem III helix was elongated by adding 10 bp just before the loop spanning nucleotides 134–139 (Figure ) (10 bases before 134 and 10 complementary bases after 139) (S3.10), by using standard site-directed mutagenesis protocols ( ). .. For in vitro transcription, the bicistronic DNA template was inserted into a pUC19 vector by using the EcoR1 (NEB) and Hind III (NEB) restriction sites.

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    New England Biolabs superhelical puc19
    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng <t>superhelical</t> <t>pUC19</t> and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.
    Superhelical Puc19, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    superhelical puc19 - by Bioz Stars, 2020-01
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    Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Journal: Frontiers in Chemistry

    Article Title: DNA oxidation profiles of copper phenanthrene chemical nucleases

    doi: 10.3389/fchem.2015.00028

    Figure Lengend Snippet: Lane 1–4 (A–D) DNA cleavage reactions with 250 nM, 500 nM, 1.0 μM, and 2.5 μM test complex (A) Cu-Phen, (B) Cu-DPQ-Phen, (C) Cu-DPPZ-Phen, and (D) Cu-Terph, 400 ng superhelical pUC19 and 1 mM added Na-L-ascorbate incubated at 37°C for 30 min . Lanes 5–16 (A–D) DNA cleavage reactions in the presence of recognition elements, methyl green (MG), netropsin (Net), and [Co(NH 3 ) 6 ]Cl 3 (Co(III)), where 400 ng pUC19 was initially pre-treated with 8 μM of respective non-covalent binding control at 37° C for 45 min and then with 250 nM, 500 nM, 1 μM, and 2.5 μM test complex in the presence of 1 mM added Na-L-ascorbate at 37°C for 30 min.

    Article Snippet: Reactions were carried out according to the following general procedure: in a total volume of 20 μL using 80 mM HEPES buffer (pH 7.2) with 25 mM NaCl, 1 mM Na-L-ascorbate, 400 ng superhelical pUC19 (NEB, N3041) and varying concentrations of test complex (250 nM, 500 nM, 1 μM and 2.5 μM).

    Techniques: Incubation, Binding Assay