ecori  (New England Biolabs)


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    Name:
    EcoRI
    Description:
    EcoRI 50 000 units
    Catalog Number:
    r0101l
    Price:
    244
    Size:
    50 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs ecori
    EcoRI
    EcoRI 50 000 units
    https://www.bioz.com/result/ecori/product/New England Biolabs
    Average 90 stars, based on 537 article reviews
    Price from $9.99 to $1999.99
    ecori - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    2) Product Images from "A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage"

    Article Title: A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky806

    ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.
    Figure Legend Snippet: ( A ) BDNPP hydrolytic cleavage mechanism in the presence of Cu 2 TPNap; ( B ) Lineweaver–Burk plot; ( C ) rate-pH profile for the cleavage of BDNPP in the presence of Cu 2 TPNap at 40°C; ( D ) DNA cleavage reactions by Cu 2 TPNap on pUC19 plasmid DNA over 1 h at 37°C in the absence of added reductant; and ( E ) T4 DNA ligase experiments with Cu 2 TPNap and restriction enzymes EcoRI and Nt.BspQI.

    Techniques Used: Plasmid Preparation

    3) Product Images from "CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection"

    Article Title: CRISPR-READI: Efficient generation of knock-in mice by CRISPR RNP Electroporation and AAV Donor Infection

    Journal: Cell reports

    doi: 10.1016/j.celrep.2019.05.103

    CRISPR-READI optimization for efficient HDR editing in mouse embryos. a Zygotes were transduced with a panel of AAV serotypes harboring a CMV-eGFP reporter and imaged by fluorescent microscopy 48 hours post-transduction. Representative embryos transduced with scAAV1-CMV-eGFP are shown (left), and mean fluorescence intensity per embryo was quantified for each serotype (right). Scale bars = 50 μm. b Cartoon depiction of CRISPR-READI workflow. Embryos are collected from superovulated female mice, transduced with rAAV1 harboring the donor template, electroporated with Cas9/sgRNA RNPs, and implanted into pseudopregnant females to generate edited mice. c Schematic of Tyr targeting strategy. The scAAV1-Tyr donor creates an EcoRI restriction site in exon 1 of the Tyr locus upon HDR editing. ITR: inverted terminal repeat, HA: homology arm, F/R: forward/reverse primers for RFLP analysis. d Optimization of rAAV1 dosage for HDR editing. Zygotes were transduced with scAAV1-Tyr at a dose of 1.1×10 8 , 4.2×10 8 , or 1.7×10 9 GCs, electroporated with RNPs 5 hours post-transduction, and then returned to rAAV1 incubation for another 19 hours. Treated embryos were cultured to the morula stage and genotyped by restriction fragment length polymorphism (RFLP) analysis (shown for dose of 1.7×10 9 GCs). Edited embryos yield 650 bp and 420 bp bands upon EcoRI digestion of the PCR amplicon (top, black arrows). HDR rate was quantified by RFLP analysis for each dose (bottom left), and embryo viability was scored as percentage of cultured embryos that reached the morula stage (bottom right). e Optimization of RNP electroporation timing relative to rAAV transduction. Zygotes were transduced with scAAV1-Tyr, electroporated at varying time points post-transduction (2, 4, 6, 8, or 10 hours), and returned to rAAV incubation for a total of 24 hours. Treated embryos were cultured to the morula stage, lysed, and assessed by RFLP analysis (right). 6 hours (*) was identified as the optimal time of RNP electroporation for maximal editing efficiency.
    Figure Legend Snippet: CRISPR-READI optimization for efficient HDR editing in mouse embryos. a Zygotes were transduced with a panel of AAV serotypes harboring a CMV-eGFP reporter and imaged by fluorescent microscopy 48 hours post-transduction. Representative embryos transduced with scAAV1-CMV-eGFP are shown (left), and mean fluorescence intensity per embryo was quantified for each serotype (right). Scale bars = 50 μm. b Cartoon depiction of CRISPR-READI workflow. Embryos are collected from superovulated female mice, transduced with rAAV1 harboring the donor template, electroporated with Cas9/sgRNA RNPs, and implanted into pseudopregnant females to generate edited mice. c Schematic of Tyr targeting strategy. The scAAV1-Tyr donor creates an EcoRI restriction site in exon 1 of the Tyr locus upon HDR editing. ITR: inverted terminal repeat, HA: homology arm, F/R: forward/reverse primers for RFLP analysis. d Optimization of rAAV1 dosage for HDR editing. Zygotes were transduced with scAAV1-Tyr at a dose of 1.1×10 8 , 4.2×10 8 , or 1.7×10 9 GCs, electroporated with RNPs 5 hours post-transduction, and then returned to rAAV1 incubation for another 19 hours. Treated embryos were cultured to the morula stage and genotyped by restriction fragment length polymorphism (RFLP) analysis (shown for dose of 1.7×10 9 GCs). Edited embryos yield 650 bp and 420 bp bands upon EcoRI digestion of the PCR amplicon (top, black arrows). HDR rate was quantified by RFLP analysis for each dose (bottom left), and embryo viability was scored as percentage of cultured embryos that reached the morula stage (bottom right). e Optimization of RNP electroporation timing relative to rAAV transduction. Zygotes were transduced with scAAV1-Tyr, electroporated at varying time points post-transduction (2, 4, 6, 8, or 10 hours), and returned to rAAV incubation for a total of 24 hours. Treated embryos were cultured to the morula stage, lysed, and assessed by RFLP analysis (right). 6 hours (*) was identified as the optimal time of RNP electroporation for maximal editing efficiency.

    Techniques Used: CRISPR, Transduction, Microscopy, Fluorescence, Mouse Assay, Incubation, Cell Culture, Polymerase Chain Reaction, Amplification, Electroporation

    4) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.
    Figure Legend Snippet: Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.

    Techniques Used: Inhibition

    ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.
    Figure Legend Snippet: ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.

    Techniques Used: Generated, Incubation

    5) Product Images from "Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine"

    Article Title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine

    Journal: Mutagenesis

    doi: 10.1093/mutage/get027

    Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).
    Figure Legend Snippet: Repair of MB + VL-induced 8-oxoG from the Ad-encoded lacZ gene in human and rodent cells measured by loss of Fpg-sensitive sites. ( A ) Southern blot analysis of the repair of MB + VL-induced 8-oxoG in the Ad lacZ gene. Shown here is a representative blot. Lanes 1 and 2 contain untreated Ad DNA, while lanes 3 and 4 contain Ad DNA exposed to 480 s VL in phosphate buffer with 20 mg/ml MB. Lanes 1–4 have not undergone any repair incubation. The presence of ssDNA breaks in the 3-kb EcoRI lacZ fragment produce smaller ssDNA fragments that migrate further than the full-length fragment. These smaller fragments appear as a smear or tail below the defined 3-kb band. Smearing below the 3-kb band in samples that have not been treated with Fpg (lanes 1 and 3) represent ssDNA breaks from other sources. It can be seen that a small amount of Fpg-sensitive 8-oxoG lesions are present prior to treatment with MB + VL (compare lanes 1 and 2). Following MB + VL exposure, a large number of Fpg-sensitive sites are generated (compare lanes 2 and 4). During repair incubation, BER removes 8-oxoG resulting in the loss of T4pdg-sensitive sites and recovery of the full-length 3-kb lacZ fragment. As long as 8-oxoG lesions persist in the lacZ DNA, Fpg will induce ssDNA breaks resulting in fewer full-length fragments and less signal compared to the control. ( B ) Quantification of the percent removal of Fpg-sensitive sites from the Ad-encoded lacZ gene in GM637F and CHO-AA8 cells. Each point on the graphs represents the arithmetic mean ± SE of the percent removal of MB + VL-induced Fpg-sensitive sites from three independent experiments. A significant increase in the percent removal of MB + VL-induced Fpg-sensitive sites was observed in GM637F at 24 h (indicated by an asterisk) and a significant difference in the percent removal of Fpg-sensitive sites was observed between GM637F and CHO-AA8 at 24 h (indicated by a cross/plus sign).

    Techniques Used: Southern Blot, Incubation, Generated

    6) Product Images from "Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System"

    Article Title: Identification of Hedysarum Varieties Using Amplified Fragment Length Polymorphism on a Capillary Electrophoresis System

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Final genotypes in a binary format for subset of the samples and alleles from the AFLP run with the primer pair FAM-Ecori-ACA and Msei-CTT as displayed in the Genotypes tab of the GeneMapper software.
    Figure Legend Snippet: Final genotypes in a binary format for subset of the samples and alleles from the AFLP run with the primer pair FAM-Ecori-ACA and Msei-CTT as displayed in the Genotypes tab of the GeneMapper software.

    Techniques Used: Software

    Related Articles

    Clone Assay:

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: .. Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: mRNA preparation and injection optn (ENSDART00000014036.10, Ensembl) and p62 (ENSDART00000140061.2, Ensembl) cDNAs were amplified from 3 dpf WT embryos by PCR (primers in ) and ligated into a vector using the Zero-blunt cloning PCR kit (450245, Invitrogen). .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Paragraph title: RNA cloning and sequencing ... An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector. .. The fl gene from the pGL3 basic plasmid (Promega Corporation, Madison, WI, USA) was amplified by PCR using the same 5′-end primer FLUCUp-EcoRI 5′-AGC ATC GAA TTC TGA GGA CGC CAA AAA CAT AAA G-3′, the 3′end primer FLUCDn-SalI 5′-CTA GTA GTC GAC AGC AAT CTT TCC GCC CTT CT-3′, and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Amplification:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: .. The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively. .. Using QuickChange II site-directed mutagenesis kit (Cat# 200523) from Agilent Technology.

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: .. Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Double stranded DNA was generated by PCR amplification with the reverse primer, and the forward primer ( 5′CAG CCA ACG GAA TTC ATA CGA CTC ACT AAA-3′ ). .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Gp140-encoded regions of env were amplified, incorporating the full native gp120 and gp41 mature protein encoding regions with the furin site replaced with SEKS and ending with amino-acid position 668, located at the membrane proximal region of gp41, followed by a His6 tag.

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: These primers amplified a single product of the expected size as confirmed by the melting temperatures of the amplicons. nptII resides in single-copy on the plasmids characterized in this study, while 16S rDNA gene resides on multiple copies on the E. coli chromosome [ ] and was used for normalization [ , , ]. .. Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C.

    Stable Transfection:

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Synthesized:

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Construct:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Real-time Polymerase Chain Reaction:

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: Paragraph title: Real-time qPCR quantification of plasmid copy number ... Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C.

    Incubation:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: .. Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. ..

    Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
    Article Snippet: Southern blotting Genomic DNA (1 μg) was separated on a 1% (w/v) agarose gel after overnight endonuclease digestion with EcoRI New England Biolabs (NEB). .. The gel was washed twice with dH2 O, incubated with the depurination solution (0.2 M HCl) for 20 min with gentle shaking and rinsed with dH2 O.

    Expressing:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: Paragraph title: 2.3. Construction of gp140 Env expression plasmids ... PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: Briefly, the monomeric DsRed expression plasmid, pDsRed-Monomer -C1 , driven by CMV enhancer/promoter, was purchased from Clontech (BD science, Inc., USA). .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Modification:

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: The human tissue plasminogen activator leader sequence was used to replace the native HIV-1 gp140 leader, a modification which has been shown to increase gp140 expression . .. PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid.

    Transformation Assay:

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. .. The DNA fragments were ligated with pCR 2.1 TOPO vector (Invitrogen), transformed into TOP10 E. coli (Invitrogen) and individual clones grown for plasmid isolation.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA).

    Activated Clotting Time Assay:

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Double stranded DNA was generated by PCR amplification with the reverse primer, and the forward primer ( 5′CAG CCA ACG GAA TTC ATA CGA CTC ACT AAA-3′ ). .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Countercurrent Chromatography:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA). .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Transfection:

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Southern Blot:

    Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
    Article Snippet: .. Southern blotting Genomic DNA (1 μg) was separated on a 1% (w/v) agarose gel after overnight endonuclease digestion with EcoRI New England Biolabs (NEB). .. The gel was washed twice with dH2 O, incubated with the depurination solution (0.2 M HCl) for 20 min with gentle shaking and rinsed with dH2 O.

    Ligation:

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: The ligation products were gel purified, phosphorylated at the 5′ end with T4 polynucleotide kinase (New England Biolabs, Beverly MA) and ligated to the 5′adaptor sequence [ 5′ TAC TAA TAC GAC TCA CT aaa 3′ (Dharmacon)]. .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA).

    RFLP Assay:

    Article Title: Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications
    Article Snippet: Paragraph title: Colony characterization using 16S rRNA gene-based RFLP assay ... Five μl of each reaction were electrophoresed on 1.5% agarose gels, and all amplicons of the expected size were individually digested with the restriction endonucleases Taq I, EcoR I and Hae III (New England Biolabs and/or Fermentas), according to the manufacturer’s suggestions.

    Generated:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: A fragment encoding UCR1 (amino acid 17-136) or UCR1C (amino acid 80-136) of human PDE4D3 was generated by PCR using GFP-PDE4D3 as a template. .. The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Double stranded DNA was generated by PCR amplification with the reverse primer, and the forward primer ( 5′CAG CCA ACG GAA TTC ATA CGA CTC ACT AAA-3′ ). .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Sequencing:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Paragraph title: RNA cloning and sequencing ... An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: A genome-wide SNP-based genetic map and QTL mapping for agronomic traits in Chinese cabbage
    Article Snippet: Paragraph title: RAD library construction and sequencing ... The DNA was digested with EcoR I (New England Biolabs, Ipswich, MA, USA), the digestion product samples were ligated to a P1 adapter with the Illumina Solexa kit ©.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: The 3′ primer incorporated a Xho I site and a His6 tag sequence followed by a stop codon. .. PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid.

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Injection:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: Paragraph title: mRNA preparation and injection ... The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Recombinant:

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Cellular Antioxidant Activity Assay:

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector. .. The fl gene from the pGL3 basic plasmid (Promega Corporation, Madison, WI, USA) was amplified by PCR using the same 5′-end primer FLUCUp-EcoRI 5′-AGC ATC GAA TTC TGA GGA CGC CAA AAA CAT AAA G-3′, the 3′end primer FLUCDn-SalI 5′-CTA GTA GTC GAC AGC AAT CTT TCC GCC CTT CT-3′, and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Molecular Cloning:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: Paragraph title: 2.3. Molecular Cloning ... The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively.

    Mutagenesis:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively. .. Using QuickChange II site-directed mutagenesis kit (Cat# 200523) from Agilent Technology.

    Isolation:

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. .. The DNA fragments were ligated with pCR 2.1 TOPO vector (Invitrogen), transformed into TOP10 E. coli (Invitrogen) and individual clones grown for plasmid isolation.

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: .. Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C. .. Real-time qPCR was conducted on a BioRad iCycler with 96-well reaction blocks in the presence of SYBR Green under the following conditions: 1X iQ SYBR Green Supermix (BioRad), 150 nM nptII (500 nM 16S) primers in a 25 μL reaction.

    Article Title: A first generation BAC-based physical map of the channel catfish genome
    Article Snippet: The DNA was isolated via an alkaline lysis method with Qiagen reagents (Qiagen, Inc., Valencia, CA) in a 96-well format using an Apricot pipettor (PerkinElmer Life and Analytical Sciences, Wellesley, MA). .. Briefly, 12 ul of DNA (approximately 400 ng of DNA) was digested with Hae III, EcoR I, Xba I, Xho I, BamH I (New England Biolabs, Ipswich, MA) in the presence of 0.1% β-mercaptoethanol and RNase DNase-free (Roche Applied Science, Indianapolis, IN) for 3 hours at 37°C in a PTC-200 thermal cycler (MJ Research, Watertown, MA).

    Size-exclusion Chromatography:

    Article Title: Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications
    Article Snippet: PCR conditions were: initial denaturing step of 95°C, for 5 min, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 55°C for 1 min and extension at 72°C for 2 min. A final extension step of 72°C for 10 min was added. .. Five μl of each reaction were electrophoresed on 1.5% agarose gels, and all amplicons of the expected size were individually digested with the restriction endonucleases Taq I, EcoR I and Hae III (New England Biolabs and/or Fermentas), according to the manufacturer’s suggestions.

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C. .. Real-time qPCR cycling was 95°C for 3 min, followed by 40 cycles of 30 sec at 95°C, 30 sec at 60°C, and 30 sec at 72°C.

    Labeling:

    Article Title: A first generation BAC-based physical map of the channel catfish genome
    Article Snippet: Briefly, 12 ul of DNA (approximately 400 ng of DNA) was digested with Hae III, EcoR I, Xba I, Xho I, BamH I (New England Biolabs, Ipswich, MA) in the presence of 0.1% β-mercaptoethanol and RNase DNase-free (Roche Applied Science, Indianapolis, IN) for 3 hours at 37°C in a PTC-200 thermal cycler (MJ Research, Watertown, MA). .. Fragments were labeled with the SNaPshot kit (Applied Biosystems, Foster City, CA) at 65°C for 60 minutes C in a PTC-200 thermal cycler (MJ Research).

    Purification:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: The ligation products were gel purified, phosphorylated at the 5′ end with T4 polynucleotide kinase (New England Biolabs, Beverly MA) and ligated to the 5′adaptor sequence [ 5′ TAC TAA TAC GAC TCA CT aaa 3′ (Dharmacon)]. .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: A genome-wide SNP-based genetic map and QTL mapping for agronomic traits in Chinese cabbage
    Article Snippet: The DNA was digested with EcoR I (New England Biolabs, Ipswich, MA, USA), the digestion product samples were ligated to a P1 adapter with the Illumina Solexa kit ©. .. The DNA was then ligated with a P2 adapter, followed by purification using 1.6 × AMPure XP Beads and then used for selective PCR using 2× Phusion PCR Master Mix (NEB) over 18 cycles.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: .. PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA).

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Polymerase Chain Reaction:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: A fragment encoding UCR1 (amino acid 17-136) or UCR1C (amino acid 80-136) of human PDE4D3 was generated by PCR using GFP-PDE4D3 as a template. .. The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively.

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: .. Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. ..

    Article Title: Exploitation of the Medfly Gut Microbiota for the Enhancement of Sterile Insect Technique: Use of Enterobacter sp. in Larval Diet-Based Probiotic Applications
    Article Snippet: PCR conditions were: initial denaturing step of 95°C, for 5 min, followed by 35 cycles of denaturation at 95°C for 45 sec, annealing at 55°C for 1 min and extension at 72°C for 2 min. A final extension step of 72°C for 10 min was added. .. Five μl of each reaction were electrophoresed on 1.5% agarose gels, and all amplicons of the expected size were individually digested with the restriction endonucleases Taq I, EcoR I and Hae III (New England Biolabs and/or Fermentas), according to the manufacturer’s suggestions.

    Article Title: A genome-wide SNP-based genetic map and QTL mapping for agronomic traits in Chinese cabbage
    Article Snippet: The DNA was digested with EcoR I (New England Biolabs, Ipswich, MA, USA), the digestion product samples were ligated to a P1 adapter with the Illumina Solexa kit ©. .. The DNA was then ligated with a P2 adapter, followed by purification using 1.6 × AMPure XP Beads and then used for selective PCR using 2× Phusion PCR Master Mix (NEB) over 18 cycles.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: .. PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA).

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The ttksr39 PCR products were purified and subjected to restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) digestion and then the purified ttksr39 insert DNAs were ligated with BamH I-XbaI digested pDsRed-Monomer-C1 vector by T4 DNA ligase (New England Biolabs, Inc., USA) generating a DsRedm-ttksr39 dual fusion reporter genetic construct. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Quantitative RT-PCR:

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. After transfection, the lentiviral particles were harvested and used to infect SUNE-1 cells, and the stably transfected cells were selected with puromycin and further confirmed with quantitative RT-PCR.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: .. The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB CatR0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively. .. Using QuickChange II site-directed mutagenesis kit (Cat# 200523) from Agilent Technology.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA). .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Silver Staining:

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino
    Article Snippet: Mthylation Sensitive Amplified Polymorphism Analysis The MSAP technique was applied to the DNA pools from three diploid lines and three tetraploid lines C. lavandulifolium They were digested with either EcoR I and Hpa II or EcoR I and Msp I (NEB) at 37 °C for 12 h. The digested fragments were ligated to 5 pmol EcoR I adaptor and 50 pmol Hpa II/Msp I adaptor by incubation with 4 U T4 DNA polymerase (NEB) at 16 °C for 4 has described for the AFLP method. .. The amplifications were electrophoresed through 10% denaturing polyacrylamide gels, running in 1× TBE buffer at 300 V for 3.5 h on ice, and then visualized by silver staining.

    Plasmid Preparation:

    Article Title: Streamlined protein expression and purification using cleavable self-aggregating tags
    Article Snippet: Materials The restriction enzymes Nde I, Hind III, EcoR I, Dpn I, Spe I and the T4 DNA ligase were from either New England Biolabs (Beverly, MA) or Takara (Dalian, China). .. The vector pTWIN1 was from New England Biolabs.

    Article Title: UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy
    Article Snippet: .. The amplified fragments were clone into KpnI/EcoRI (for Cerulean-UCR1) and EcoRV/EcoRI (for Cerulean-UCR1C) sites of FLAG-Cerulean vector digested with restriction enzymes EcoRI (NEB Cat# R0101S)/KpnI (NEB Cat#R0142) (for cerulean-UCR1), and EcoRI/EcoRV (NEB Cat# R0195S) (for Cerulean-UCR1C) respectively. .. Using QuickChange II site-directed mutagenesis kit (Cat# 200523) from Agilent Technology.

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. .. The DNA fragments were ligated with pCR 2.1 TOPO vector (Invitrogen), transformed into TOP10 E. coli (Invitrogen) and individual clones grown for plasmid isolation.

    Article Title: Comparison of Neutralizing Antibody Responses Elicited from Highly Diverse Polyvalent Heterotrimeric HIV-1 gp140 Cocktail Immunogens versus a Monovalent Counterpart in Rhesus Macaques
    Article Snippet: .. PCR products were digested with EcoR I (New England Biolabs, Beverly, MA; NEB) and Xho I (NEB), gel purified (Qiagen, Venlo, The Netherlands, and ligated into the pLEXm plasmid. .. Ligation reactions were performed at room temperature (RT) for 1 hr using T4 DNA ligase (NEB) prior to transformation into DH5 alpha Escherichia coli cells (Invitrogen, Carlsbad, CA).

    Article Title: DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair
    Article Snippet: Paragraph title: Enzymatic digestion of plasmid and total genomic DNA ... EcoR I (New England Biolabs, Ipswich, MA) that has a single restriction site in pBR322 DNA was used to generate its linear forms.

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: Paragraph title: Real-time qPCR quantification of plasmid copy number ... Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector. .. The fl gene from the pGL3 basic plasmid (Promega Corporation, Madison, WI, USA) was amplified by PCR using the same 5′-end primer FLUCUp-EcoRI 5′-AGC ATC GAA TTC TGA GGA CGC CAA AAA CAT AAA G-3′, the 3′end primer FLUCDn-SalI 5′-CTA GTA GTC GAC AGC AAT CTT TCC GCC CTT CT-3′, and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA).

    Article Title: MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma
    Article Snippet: .. Generation of stably transfected cell lines The sequence of pri-miR-145 was synthesized from human genomic DNA using PCR, and cloned into retroviral vector pMSCV-puromycin with Bgl II and EcoR I (New England Biolab). .. A plasmid mixture containing pMSCV-miR-24 or empty pMSCV vector, along with retroviral packaging vector PIK, was co-transfected into 293FT cells.

    Software:

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C. .. Threshold cycles (Ct) were determined with iCycler (BioRad) software for all samples.

    SYBR Green Assay:

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C. .. Real-time qPCR was conducted on a BioRad iCycler with 96-well reaction blocks in the presence of SYBR Green under the following conditions: 1X iQ SYBR Green Supermix (BioRad), 150 nM nptII (500 nM 16S) primers in a 25 μL reaction.

    Functional Assay:

    Article Title: Role of Novel Multidrug Efflux Pump Involved in Drug Resistance in Klebsiella pneumoniae
    Article Snippet: Cloning of kpnGH in Hyper Susceptible E. coli Strain KAM32 The putative efflux genes kpnG and kpnH were amplified by standard PCR protocol using primers kpnG -F; kpnG -R and kpnH -F; kpnH -R ( ) respectively and cloned into EcoR I and Pst I (New England Biolabs, MA, USA) site of pUC18. .. The resulting recombinant plasmid pkpnG , pkpnH and pkpnGH were transformed into E. coli KAM32 (ΔacrAB and ΔydhE ) for functional characterization.

    Agarose Gel Electrophoresis:

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted. ..

    Article Title: Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection
    Article Snippet: .. Southern blotting Genomic DNA (1 μg) was separated on a 1% (w/v) agarose gel after overnight endonuclease digestion with EcoRI New England Biolabs (NEB). .. The gel was washed twice with dH2 O, incubated with the depurination solution (0.2 M HCl) for 20 min with gentle shaking and rinsed with dH2 O.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The pDsRed-Monomer-C1 digested with restriction enzyme of BamH I and Xba I (New England Biolabs, Inc., USA) was purified from 1% agarose gel by PCR/Gel Extraction kit (Geneaid Inc., Taiwan) and used as cloning vector. .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    In Vitro:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Transgenic Assay:

    Article Title: BglBrick vectors and datasheets: A synthetic biology platform for gene expression
    Article Snippet: In order to determine plasmid copy number (i.e. number of plasmids per genomic equivalent), E. coli DH1 and BLR transgenic strains with a single nptII integration (data not shown) were used for calibration. .. Total DNA isolated from each strain was first digested overnight using EcoR I (New England Biolabs) at 37°C.

    Produced:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: The sequence was confirmed by Sanger sequencing (BaseClear, Netherlands), after which optn and p62 cDNAs were subcloned into a pCS2+ expression vector. optn ΔUBAN cDNA was produced by in vitro transcription of optn -pCS2+ constructs digested by Sca1(R3122, NEB), which excludes the region encoding the UBAN protein domain. optn ΔLIR cDNA was amplified from optn -pCS2+ constructs by designed primers , excluding the LIR protein domain. .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation.

    Alkaline Lysis:

    Article Title: A first generation BAC-based physical map of the channel catfish genome
    Article Snippet: The DNA was isolated via an alkaline lysis method with Qiagen reagents (Qiagen, Inc., Valencia, CA) in a 96-well format using an Apricot pipettor (PerkinElmer Life and Analytical Sciences, Wellesley, MA). .. Briefly, 12 ul of DNA (approximately 400 ng of DNA) was digested with Hae III, EcoR I, Xba I, Xho I, BamH I (New England Biolabs, Ipswich, MA) in the presence of 0.1% β-mercaptoethanol and RNase DNase-free (Roche Applied Science, Indianapolis, IN) for 3 hours at 37°C in a PTC-200 thermal cycler (MJ Research, Watertown, MA).

    BAC Assay:

    Article Title: A first generation BAC-based physical map of the channel catfish genome
    Article Snippet: Paragraph title: BAC library fingerprinting ... Briefly, 12 ul of DNA (approximately 400 ng of DNA) was digested with Hae III, EcoR I, Xba I, Xho I, BamH I (New England Biolabs, Ipswich, MA) in the presence of 0.1% β-mercaptoethanol and RNase DNase-free (Roche Applied Science, Indianapolis, IN) for 3 hours at 37°C in a PTC-200 thermal cycler (MJ Research, Watertown, MA).

    CTG Assay:

    Article Title: Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism
    Article Snippet: Synthesis of cDNA from adaptor-ligated RNA was accomplished using the Thermoscript kit (Invitrogen) and the reverse primer ( 5′ GAC TAG CTG GAA TTC AAG GAT GCG GTT AAA-3′ ). .. An aliquot of the PCR products was digested with EcoR I (New England Biolabs) and ligated to form concatemers that were resolved by agarose gel electrophoresis and extracted.

    Article Title: Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging
    Article Snippet: The truncated tksr39 gene was amplified from the plasmid pttksr39 (generous gifts from Professor FD Chen, TransWorld University, Taiwan) by polymerase chain reaction (PCR) with 5′-end primer ttkUp/BamHI (5′-CAA GAC GGA TCC TCT GGT AAA ATG CCC ACG CTA CTG C-3′), 3′-end primer ttkDn-XbaI (5′-GTA TTC TCT AGA TCA GTT AGC CTC CCC CAT C-3′), and the proof-reading KOD Taq DNA polymerase (Novagen Inc., USA). .. The DsRedm-ttksr39 plasmid was digested with restriction enzyme of EcoR I and Sal I (New England Biolabs, Inc., USA) and used for a cloning vector.

    Gel Extraction:

    Article Title: The selective autophagy receptors Optineurin and p62 are both required for zebrafish host resistance to mycobacterial infection
    Article Snippet: .. The PCR products were gel purified by Quick gel Extraction PCR Purification Combo Kit (K220001, Invitrogen) and the two fragments and pCS2+ plasmid were digested by BamH1(R0136S, NEB) and EcoR1(R0101S, NEB), after which the two fragments were ligated into pCS2+ plasmid by T4 DNA ligase. p62 ΔUBA cDNA was obtained from a p62 -pCS2+ construct by Nco1(R0193S, NEB) digestion and religation, which excludes the region encoding the UBA protein domain. p62 ΔLIR cDNA was obtained from a p62 -pCS2+ construct by NcoN1 digestion and religation. .. Optn mRNA, optn ΔUBAN, and optn ΔLIR mRNA was generated using SP6 mMessage mMachine kit (Life Technologies) from Kpn1 or Sac1(R0156S, NEB) digested optn –pCS2+ constructs.

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