kpni  (New England Biolabs)


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  • 99
    Name:
    NheI
    Description:
    NheI 5 000 units
    Catalog Number:
    R0131L
    Price:
    277
    Size:
    5 000 units
    Category:
    Restriction Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    New England Biolabs kpni
    NheI
    NheI 5 000 units
    https://www.bioz.com/result/kpni/product/New England Biolabs
    Average 99 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    kpni - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys"

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

    Journal:

    doi: 10.1128/AEM.02346-06

    PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
    Figure Legend Snippet: PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Techniques Used:

    PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates
    Figure Legend Snippet: PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Techniques Used: Labeling

    PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics
    Figure Legend Snippet: PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Techniques Used:

    PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.
    Figure Legend Snippet: PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Techniques Used:

    2) Product Images from "Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine"

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    Journal: Tropical Medicine and Infectious Disease

    doi: 10.3390/tropicalmed2030037

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.
    Figure Legend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Techniques Used: Recombinant

    3) Product Images from "Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid"

    Article Title: Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid

    Journal:

    doi: 10.1128/JB.00939-08

    PFGE of type B isolates. Agarose plugs containing DNA from each specified isolate were digested with KpnI (A) or ApaI (B) and then subjected to PFGE and staining with ethidium bromide. Numbers at right of each blot indicate migration of size markers in
    Figure Legend Snippet: PFGE of type B isolates. Agarose plugs containing DNA from each specified isolate were digested with KpnI (A) or ApaI (B) and then subjected to PFGE and staining with ethidium bromide. Numbers at right of each blot indicate migration of size markers in

    Techniques Used: Staining, Migration

    4) Product Images from "RNA aptamer inhibitors of a restriction endonuclease"

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv702

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.
    Figure Legend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Techniques Used: In Vitro, Selection

    5) Product Images from "EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide - diamino hexane) for the delivery of therapeutic pCMV-RAE-1? to pancreatic islets"

    Article Title: EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide - diamino hexane) for the delivery of therapeutic pCMV-RAE-1? to pancreatic islets

    Journal:

    doi: 10.1016/j.jconrel.2011.10.022

    Digestion of pCMV-RAE-1γ with KpnI and NotI.
    Figure Legend Snippet: Digestion of pCMV-RAE-1γ with KpnI and NotI.

    Techniques Used:

    6) Product Images from "Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection"

    Article Title: Restriction Endonucleases from Invasive Neisseria gonorrhoeae Cause Double-Strand Breaks and Distort Mitosis in Epithelial Cells during Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0114208

    Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).
    Figure Legend Snippet: Lysates of N. gonorrhoeae fragments pECFP-N1 and damage DNA from VK2/E6E7 cells. A. DNA agarose gel showing the digestion of pECFP-N1 plasmid by HindIII (positive control, lane 2), MS11 P+ lysate (lane 3), and MS11 P+ HI lysate (lane 5). Lane 5 shows bacterial MS11 P+ lysate without pECFP-N1 and lane 1 shows uncut circular pECFP-N1. B. PFGE analysis of purified VK2/E6E7 genomic DNA treated for 24 h with: lane 1: PBS (negative control), lane 2: MS11 P+ lysate, lane 3: MS11 P+ HI lysate. Lane 4 shows bacterial MS11 P+ lysate without VK2/E6E7 genomic DNA. C. Graph showing quantification of DNA smears (measured directly underneath and below the band). Shown are smear pixel intensities of cellular DNA alone and cellular DNA exposed to bacterial lysates and HI bacterial lysates. D. PFGE showing genomic DNA subjected to commercial restriction enzymes for 24 h. Lane 1: DNA incubated with CutSmart reaction buffer (negative control). Lane 2: DNA incubated with NgoMIV. Lane 3: DNA incubated with MfeI, Lane 4: DNA incubated with NgoMIV and MfeI Lane 5: DNA incubated with NgoMIV and BamHI/KpnI/MfeI (BKM).

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Positive Control, Purification, Negative Control, Incubation

    7) Product Images from "The Polyphosphate Kinase Gene ppk2 Is Required for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence"

    Article Title: The Polyphosphate Kinase Gene ppk2 Is Required for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence

    Journal: mBio

    doi: 10.1128/mBio.00039-13

    Complementation of ppk2 ::Tn. (A) RT-PCR analysis of wild-type M . tuberculosis CDC 1551 expression of intergenic regions from MT3331 to desA3/MT3326 during late log phase. Chromosomal DNA was used as a control. Primer sets 1 to 5 ( Table 1 ) target specific intergenic areas, as indicated in the figure; primer set 6 targets ppk2 . (B) PCR analysis of genomic DNA from the wild-type, ppk2 ::Tn, and ppk2 ::Tn Comp strains. Primer set A targets the ppk2 gene (bp −50 to 430), yielding a 480-bp product in the wild type, a 2,547-bp product in the ppk2 ::Tn mutant, and both products in the ppk2 ::Tn Comp strain. Primer set B targets bp 108 to 661 of ppk2 , yielding a 553-bp product in all strains. (C) Southern hybridization demonstrating binding of the ppk2 -specific probe to KpnI-digested fragments of the expected size in the wild type (1,669 bp), ppk2 ::Tn mutant (2,266 bp), and ppk2 ::Tn Comp strain (2,266 and 6,095 bp). (D) RT-PCR analysis of ppk2 expression in the wild-type, ppk2 ::Tn, and ppk2 ::Tn Comp strains during early stationary phase of growth.
    Figure Legend Snippet: Complementation of ppk2 ::Tn. (A) RT-PCR analysis of wild-type M . tuberculosis CDC 1551 expression of intergenic regions from MT3331 to desA3/MT3326 during late log phase. Chromosomal DNA was used as a control. Primer sets 1 to 5 ( Table 1 ) target specific intergenic areas, as indicated in the figure; primer set 6 targets ppk2 . (B) PCR analysis of genomic DNA from the wild-type, ppk2 ::Tn, and ppk2 ::Tn Comp strains. Primer set A targets the ppk2 gene (bp −50 to 430), yielding a 480-bp product in the wild type, a 2,547-bp product in the ppk2 ::Tn mutant, and both products in the ppk2 ::Tn Comp strain. Primer set B targets bp 108 to 661 of ppk2 , yielding a 553-bp product in all strains. (C) Southern hybridization demonstrating binding of the ppk2 -specific probe to KpnI-digested fragments of the expected size in the wild type (1,669 bp), ppk2 ::Tn mutant (2,266 bp), and ppk2 ::Tn Comp strain (2,266 and 6,095 bp). (D) RT-PCR analysis of ppk2 expression in the wild-type, ppk2 ::Tn, and ppk2 ::Tn Comp strains during early stationary phase of growth.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Mutagenesis, Hybridization, Binding Assay

    Related Articles

    Clone Assay:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: For both PCR reactions, iProof High-Fidelity DNA Polymerase (Bio-Rad) was used to avoid the introduction of mutations. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA). .. The plasmid with the ancestral allele (C) was generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using primer pair 5′-GCCCTTGACAAGCT C ACAAACTTGTAGGAGGG-3′ and 5′-CCCTCCTACAAGTTTGT G AGCTTGTCAAGGGC-3′ (target in bold) according to the manufacturer's recommendations.

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: The N297A mutant was similarly constructed from the same vector using the internal mismatched primer mut-3:5′-CTCGTCATGCGGTCGTGCATG -3′ and its complement to incorporate an AAC to GCC substitution and Fcmut-1:5′-ACCCTGCTTGCTCAACTCT-3′ and Fcmut-1:3′-TTGATGAGTTTGGACAAACCA-5′ as flanking primers. .. PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. To verify incorporation of the desired mutation and to check for PCR-induced errors, the entire coding sequence of the new expression plasmid was sequenced on both strands.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified.

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: In brief, a region of 450 bp of human SETX cDNA was PCR amplified from the 3′ end of senataxin using Ab-1F/Ab-1R primer pair. .. This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. A second region of 1.1 kb of SETX was PCR amplified from the 5′ end of senataxin using Ab-2F/Ab-2R primer pair.

    Article Title: A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease
    Article Snippet: SOFA-HDV-Rz cleavage assays were performed in the presence of 1 pmol of either 3′- (NS substrate) or internally 32 P-labelled target mRNA (all other substrates) mixed with SOFA-HDV-Rz (20 pmol) in a 20 µL mixture containing 50 mM Tris-HCl, pH 7.5, and 10 mM MgCl2 and then incubated at 37°C for 2 h. The reactions were stopped by the addition of denaturing loading buffer, separated on denaturing 5% PAGE gels, and then analyzed by autoradiography on a Phosphorscreen (GE Healthcare). .. The tet-responsive Pol II promoter Ptight was amplified from the plasmid pTRE-Tight (Clontech Laboratories, Inc.), cloned in pGEM-T (Promega) and subcloned in pBudCE4.1 (Invitrogen) with the added restriction sites BbsI and NheI (NEB). .. The Pol III promoter PhHI was amplified from the plasmid psiRNA-hH1GFPzeo (InvivoGen) and cloned in the engineered plasmid with the added restriction sites BspHI and SbfI.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant Sm MTAP ... The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: The full length PSMB5 from CMY (WT) and CMY-BR200 underwent PCR purification clean up (Qiagen). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

    Article Title: A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)
    Article Snippet: In this investigation, the published sequences of human MPDU1 at the genomic level (accession number ) and of the coding region (accession number XM_008236) have been used and confirmed. .. The cDNA generated by the PCR reaction described above using SL35-F-(13F) and SL35-R-(13R) was digested with NheI (New England BioLabs Inc.) and cloned into the expression vector pTre2hyg (CLONTECH Laboratories Inc., Palo Alto, California, USA) that had been digested with NheI and EcoRV (New England BioLabs Inc.). .. After amplification in bacteria (TOP10F′; Invitrogen Corp., San Diego, California, USA), the vector constructs were purified, and the sequence of the coding region was verified by direct sequencing using a LI-COR sequencer (LI-COR Biosciences, Bad Homburg, Germany).

    Article Title: Transient AID expression for in situ mutagenesis with improved cellular fitness
    Article Snippet: Site directed mutagenesis using QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to generate the AID S38A mutant with compromised activity and AID-M6A with total loss of activity . .. Wild type AID and mutants AID genes were cloned into a lentiviral vector pAS3w.Ppuro by the HpaI and NheI restriction sites (New England BioLabs, Ipswich, MA, USA). .. An HA tag epitope (YPYDVPDYA) was fused to the C-terminus of AID and AID mutants to allow detection by immunoblot analysis.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: This CFA MEFA chimeric gene, after silent mutation to fit PCR primer design and cloning purposes, was synthesized (Integrated DNA Technologies, Inc., Coralville, IA). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    Amplification:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA). .. Following agarose gel electrophoresis, DNA fragments of 200–800 base pairs (bp) were isolated from the gel and ligated to an adapter SNX/SNX rev linker [ ].

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: A 3.7 kb segment containing the derived T allele of rs11868112 and the putative RAR binding site (see ) was amplified by nested PCR. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The sequences of primers for Fgf15 promoter: forward, 5′-gatcGCTAGCGCTGTCTTCAGACACAC-3′ and reverse, 5′-GCTCGGGCCACCGCACCTCGAGgatc-3′.

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site. .. Primer NdeI-KO contained one silent nucleotide change (underlined) to inactivate the NdeI site in the PCR fragment.

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: In brief, a region of 450 bp of human SETX cDNA was PCR amplified from the 3′ end of senataxin using Ab-1F/Ab-1R primer pair. .. This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain.

    Article Title: A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease
    Article Snippet: SOFA-HDV-Rz cleavage assays were performed in the presence of 1 pmol of either 3′- (NS substrate) or internally 32 P-labelled target mRNA (all other substrates) mixed with SOFA-HDV-Rz (20 pmol) in a 20 µL mixture containing 50 mM Tris-HCl, pH 7.5, and 10 mM MgCl2 and then incubated at 37°C for 2 h. The reactions were stopped by the addition of denaturing loading buffer, separated on denaturing 5% PAGE gels, and then analyzed by autoradiography on a Phosphorscreen (GE Healthcare). .. The tet-responsive Pol II promoter Ptight was amplified from the plasmid pTRE-Tight (Clontech Laboratories, Inc.), cloned in pGEM-T (Promega) and subcloned in pBudCE4.1 (Invitrogen) with the added restriction sites BbsI and NheI (NEB). .. The Pol III promoter PhHI was amplified from the plasmid psiRNA-hH1GFPzeo (InvivoGen) and cloned in the engineered plasmid with the added restriction sites BspHI and SbfI.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: The RT product was used as a template for PCR; after adenylation, the amplification product was cloned into the pTZ57R/T vector and transformed into Escherichia coli DH5α cells. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways
    Article Snippet: The template clone of STAT3 (BC014482) was obtained from transOMIC technologies (Huntsville, AL, USA), and amplified the template using the following primer: forward, 5′-TGCTAGC GGACCCCTGATTTTAGCA-3′; reverse, 5′-GCTCGA GGGAACCACAAAGTTAGTAGTTT-3′. .. The PCR product was digested by NheI and XhoI restriction enzymes (NEB), and then the product was ligated into the same sites of pcDNA3.1 ( − ) expression vector.

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: To clone the PSMB5 subunit, RNA was isolated from CMY-BR200 and from the parental CMY cell lines using RNeasy mini kit (Qiagen, Gaithersburg, MD). cDNA was prepared by reverse transcription using iScript™ (Bio-Rad) and whole length PSMB5 was amplified by PCR using the following primer set: Forward: 5′-ATTA GCTAGC AGACATGGCGCTTGCCAGCGTGTT-3′ containing the NheI restriction site (GCTAGC), and Reverse: 5′-TATA CTCGAG TCAGGGGGTAGAGCCACTATACTTCT-3′ containing the XhoI restriction site (CTCGAG) (LifeTechnologies). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The sequence of a ∼550-bp Xenopus rhodopsin promoter, characterized previously, was amplified by PCR to include NdeI, PmeI, and I-SceI sites at the 5′ end and an NheI site at the 3′ end. pXOP-SCFP3A-N1 and pXOP-SYFP2-N1 were generated using pSCFP3A-N1 and pSYFP2-N1. .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: TRPV5N358Q was obtained by in vitro mutagenesis of TRPV5-pCINeo/IRES-GFP cDNA according to the manufacturer's instructions (Stratagene, La Jolla, CA). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites. .. All constructs were verified by DNA sequence analysis.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The synthetic CFA MEFA gene was amplified in a PCR with Pfu Taq polymerase (Strategene, La Jolla, CA) and primers pETCFA-F (5′-GTGAGT GCTAGC GCAGTAGAGGATTTTTTCATT-3′ [NheI site underlined]) and pETCFA-R (5′-CTCT CGGCCG TTATCAGGCTCCCAAAGTCATTACAAG-3′ [EagI site underlined]). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    Synthesized:

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: From the total mRNA from the adult worm, strand cDNAs were synthesized by RT-PCR employing the SuperScript III First-Strand Synthesis System from Promega. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: This CFA MEFA chimeric gene, after silent mutation to fit PCR primer design and cloning purposes, was synthesized (Integrated DNA Technologies, Inc., Coralville, IA). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    Construct:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: A partial genomic library enriched in GT repeats was constructed using the procedures described by Hamilton et al. [ ]. .. Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA).

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: The N297A mutant was similarly constructed from the same vector using the internal mismatched primer mut-3:5′-CTCGTCATGCGGTCGTGCATG -3′ and its complement to incorporate an AAC to GCC substitution and Fcmut-1:5′-ACCCTGCTTGCTCAACTCT-3′ and Fcmut-1:3′-TTGATGAGTTTGGACAAACCA-5′ as flanking primers. .. PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified.

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: An HXB2 molecular clone (pHXB2AF) was used to construct a molecular deletion clone lacking the integrase coding region. pHXB2AF is derived from pHXB2WT [ ], which expresses the full length HIV-1 sequence HXB2 (9719 bp, Genbank accession number ), with all bacterial sequences non-essential for bacterial expression and replication removed. .. Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site.

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. The 5′ SETX fragment was cloned into EcoRI and NotI sites of pGEX-5X-1 plasmid (GE Healthcare), containing an N-terminal GST tag.

    Article Title: Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells
    Article Snippet: Following plasmids were generous gifts from Prof. Sanjeev Galande (IISER Pune, India): RFP tagged constructs of full length β-catenin (β-cat FL-RFP), N terminal β-catenin (β-cat N-RFP), C- terminal β-catenin (β-cat C-RFP), GST tagged full-length β-catenin (β-cat FL-GST), Flag tagged TCF4 construct (Flag-TCF4) and Flag tagged Ctbp1 plasmid construct (Flag-Ctbp1) were kind gifts from Prof. G. Chinnadurai (Saint Louis University, School of Medicine). .. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs.

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The vectors pXOP-SCFP3A-N1, pXOP-SYFP2-N1, and pXOP-SYFP2-SCFP3A were constructed for use in the generation of transgenic Xenopus laevis . .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: TRPV5N358Q was obtained by in vitro mutagenesis of TRPV5-pCINeo/IRES-GFP cDNA according to the manufacturer's instructions (Stratagene, La Jolla, CA). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites. .. All constructs were verified by DNA sequence analysis.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The CFA/I major structural subunit CfaB gene was used as the backbone to construct the CFA MEFA gene. .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    CTL Assay:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: Knockdown of Klf15 was achieved using a Klf15 shRNA construct (shKlf15 ) or empty control virus shRNA (Ctl ) as previously described . .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Nested PCR:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: A 3.7 kb segment containing the derived T allele of rs11868112 and the putative RAR binding site (see ) was amplified by nested PCR. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Luciferase:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: Paragraph title: Luciferase reporter gene assays ... After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The sequences of primers for Fgf15 promoter: forward, 5′-gatcGCTAGCGCTGTCTTCAGACACAC-3′ and reverse, 5′-GCTCGGGCCACCGCACCTCGAGgatc-3′.

    Activity Assay:

    Article Title: Transient AID expression for in situ mutagenesis with improved cellular fitness
    Article Snippet: Site directed mutagenesis using QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to generate the AID S38A mutant with compromised activity and AID-M6A with total loss of activity . .. Wild type AID and mutants AID genes were cloned into a lentiviral vector pAS3w.Ppuro by the HpaI and NheI restriction sites (New England BioLabs, Ipswich, MA, USA).

    Expressing:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: An HXB2 molecular clone (pHXB2AF) was used to construct a molecular deletion clone lacking the integrase coding region. pHXB2AF is derived from pHXB2WT [ ], which expresses the full length HIV-1 sequence HXB2 (9719 bp, Genbank accession number ), with all bacterial sequences non-essential for bacterial expression and replication removed. .. Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site.

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: Paragraph title: Cloning and expression of senataxin antigens ... This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant Sm MTAP ... The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways
    Article Snippet: The template clone of STAT3 (BC014482) was obtained from transOMIC technologies (Huntsville, AL, USA), and amplified the template using the following primer: forward, 5′-TGCTAGC GGACCCCTGATTTTAGCA-3′; reverse, 5′-GCTCGA GGGAACCACAAAGTTAGTAGTTT-3′. .. The PCR product was digested by NheI and XhoI restriction enzymes (NEB), and then the product was ligated into the same sites of pcDNA3.1 ( − ) expression vector. .. The MCF-7 cells were transfected with the pcDNA3.1-STAT3 plasmid and pcDNA3.1 control vector by using TransIT® -2020 transfection reagent, according to the manufacturer's instructions (Mirus Bio, Inc., Madison, WI, USA).

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The anti-Env b12 Fab fragment recombinant expression vector was a kind gift from Dr. Dennis Burton . .. The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. Expression of b12 was carried out in Escherichia coli XL1 Blue competent cells (Stratagene; San Diego, CA) as previously described .

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: These expression vectors were used to transfect HEK293T cells. .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence.

    Article Title: A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)
    Article Snippet: In this investigation, the published sequences of human MPDU1 at the genomic level (accession number ) and of the coding region (accession number XM_008236) have been used and confirmed. .. The cDNA generated by the PCR reaction described above using SL35-F-(13F) and SL35-R-(13R) was digested with NheI (New England BioLabs Inc.) and cloned into the expression vector pTre2hyg (CLONTECH Laboratories Inc., Palo Alto, California, USA) that had been digested with NheI and EcoRV (New England BioLabs Inc.). .. After amplification in bacteria (TOP10F′; Invitrogen Corp., San Diego, California, USA), the vector constructs were purified, and the sequence of the coding region was verified by direct sequencing using a LI-COR sequencer (LI-COR Biosciences, Bad Homburg, Germany).

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The synthetic CFA MEFA gene was amplified in a PCR with Pfu Taq polymerase (Strategene, La Jolla, CA) and primers pETCFA-F (5′-GTGAGT GCTAGC GCAGTAGAGGATTTTTTCATT-3′ [NheI site underlined]) and pETCFA-R (5′-CTCT CGGCCG TTATCAGGCTCCCAAAGTCATTACAAG-3′ [EagI site underlined]). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ). .. The cloned CFA MEFA chimeric gene was verified first with DNA sequencing.

    Transformation Assay:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA). .. For enrichment, the DNA was denatured, hybridized to biotin-labeled repeat sequence (GT)10 probes and then attached to streptavidin-coated magnetic beads (Promega, Madison, WI, USA).

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. The 5′ SETX fragment was cloned into EcoRI and NotI sites of pGEX-5X-1 plasmid (GE Healthcare), containing an N-terminal GST tag.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: The RT product was used as a template for PCR; after adenylation, the amplification product was cloned into the pTZ57R/T vector and transformed into Escherichia coli DH5α cells. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Template plasmid DNA was digested with DpnI prior to purification of the mutant plasmid by QIAquick PCR Purification Kit (QIAgen, Inc.) and subsequent transformation. .. The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs).

    Over Expression:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: Adenoviral overexpression studies in mouse primary small intestinal epithelial cells were performed using an adenoviral construct carrying the mouse Klf15 gene (adKlf15 ) or empty control virus (Ctl ) as previously described . .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. The 5′ SETX fragment was cloned into EcoRI and NotI sites of pGEX-5X-1 plasmid (GE Healthcare), containing an N-terminal GST tag.

    Derivative Assay:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: A 3.7 kb segment containing the derived T allele of rs11868112 and the putative RAR binding site (see ) was amplified by nested PCR. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: An HXB2 molecular clone (pHXB2AF) was used to construct a molecular deletion clone lacking the integrase coding region. pHXB2AF is derived from pHXB2WT [ ], which expresses the full length HIV-1 sequence HXB2 (9719 bp, Genbank accession number ), with all bacterial sequences non-essential for bacterial expression and replication removed. .. Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site.

    Transfection:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA). .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. To verify incorporation of the desired mutation and to check for PCR-induced errors, the entire coding sequence of the new expression plasmid was sequenced on both strands.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified.

    Article Title: Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells
    Article Snippet: Plasmids were used for transient transfection in Gc1-Spg cells at a final concentration of 1.5 μg/ml using Lipofectamine 2000 as per manufacturer's instructions. .. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs.

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways
    Article Snippet: Paragraph title: Plasmid construction and transfection ... The PCR product was digested by NheI and XhoI restriction enzymes (NEB), and then the product was ligated into the same sites of pcDNA3.1 ( − ) expression vector.

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: Paragraph title: Cloning, DNA sequencing and transfection ... PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Sequencing:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Paragraph title: 4.2. Library Construction and Sequencing ... Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA).

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: An HXB2 molecular clone (pHXB2AF) was used to construct a molecular deletion clone lacking the integrase coding region. pHXB2AF is derived from pHXB2WT [ ], which expresses the full length HIV-1 sequence HXB2 (9719 bp, Genbank accession number ), with all bacterial sequences non-essential for bacterial expression and replication removed. .. Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site.

    Article Title: A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease
    Article Snippet: The tet-responsive Pol II promoter Ptight was amplified from the plasmid pTRE-Tight (Clontech Laboratories, Inc.), cloned in pGEM-T (Promega) and subcloned in pBudCE4.1 (Invitrogen) with the added restriction sites BbsI and NheI (NEB). .. The different SOFA-HDV-Rz were PCR amplified and inserted after the PhHI promoter of pDUAL-JU using added BbsI restriction sites.

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The sequence of a ∼550-bp Xenopus rhodopsin promoter, characterized previously, was amplified by PCR to include NdeI, PmeI, and I-SceI sites at the 5′ end and an NheI site at the 3′ end. pXOP-SCFP3A-N1 and pXOP-SYFP2-N1 were generated using pSCFP3A-N1 and pSYFP2-N1. .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence. .. The SV40 early polyadenylation signal sequence present in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with NotI and AflII (New England Biolabs, Ipswich, Massachusetts) and replaced by the SV40 late polyadenylation signal sequence.

    Article Title:
    Article Snippet: The Dye Terminator Cycle Sequencing FS Ready Reaction kit was from Applied Biosystems. .. NheI, BstApI, ApaI, BsaWI, BbsI, and HindIII were obtained from New England Biolabs (Ipswich, MA).

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: Epitopes from CS1 to -6 were inserted in a sequence so that the resulting CFA MEFA has similar antigenic propensity to the major subunit CfaB. .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    Ligation:

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The anti-Env b12 Fab fragment recombinant expression vector was a kind gift from Dr. Dennis Burton . .. The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. Expression of b12 was carried out in Escherichia coli XL1 Blue competent cells (Stratagene; San Diego, CA) as previously described .

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Error prone PCR (epPCR) was performed using the GeneMorph II Random Mutagenesis kit (Stratagene, Inc.) using 25 replication cycles and 10 ng/μL template plasmid. .. The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs). .. Ligations were performed for 15 hours at 4 °C using T4 DNA ligase (Promega, Inc.) and contained 3:1 insert:vector ratio.

    Generated:

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. CHO-K1 cells (European Collection of Cell Cultures) were transfected with plasmid using FuGene (Promega) and positive clones selected, expanded and purified as previously described for hexa-Fc .

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The sequence of a ∼550-bp Xenopus rhodopsin promoter, characterized previously, was amplified by PCR to include NdeI, PmeI, and I-SceI sites at the 5′ end and an NheI site at the 3′ end. pXOP-SCFP3A-N1 and pXOP-SYFP2-N1 were generated using pSCFP3A-N1 and pSYFP2-N1. .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: The pCINeo/IRES-GFP plasmid encoding TRPV5 was generated as described previously ( ). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites.

    Article Title: A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)
    Article Snippet: In this investigation, the published sequences of human MPDU1 at the genomic level (accession number ) and of the coding region (accession number XM_008236) have been used and confirmed. .. The cDNA generated by the PCR reaction described above using SL35-F-(13F) and SL35-R-(13R) was digested with NheI (New England BioLabs Inc.) and cloned into the expression vector pTre2hyg (CLONTECH Laboratories Inc., Palo Alto, California, USA) that had been digested with NheI and EcoRV (New England BioLabs Inc.). .. After amplification in bacteria (TOP10F′; Invitrogen Corp., San Diego, California, USA), the vector constructs were purified, and the sequence of the coding region was verified by direct sequencing using a LI-COR sequencer (LI-COR Biosciences, Bad Homburg, Germany).

    DNA Sequencing:

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Paragraph title: Genomic library construction, screening, and DNA sequencing ... Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified.

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: Paragraph title: Cloning, DNA sequencing and transfection ... PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Polymerase Chain Reaction:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA). .. Following agarose gel electrophoresis, DNA fragments of 200–800 base pairs (bp) were isolated from the gel and ligated to an adapter SNX/SNX rev linker [ ].

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: For both PCR reactions, iProof High-Fidelity DNA Polymerase (Bio-Rad) was used to avoid the introduction of mutations. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The sequences of primers for Fgf15 promoter: forward, 5′-gatcGCTAGCGCTGTCTTCAGACACAC-3′ and reverse, 5′-GCTCGGGCCACCGCACCTCGAGgatc-3′.

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: The N297A mutant was similarly constructed from the same vector using the internal mismatched primer mut-3:5′-CTCGTCATGCGGTCGTGCATG -3′ and its complement to incorporate an AAC to GCC substitution and Fcmut-1:5′-ACCCTGCTTGCTCAACTCT-3′ and Fcmut-1:3′-TTGATGAGTTTGGACAAACCA-5′ as flanking primers. .. PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. To verify incorporation of the desired mutation and to check for PCR-induced errors, the entire coding sequence of the new expression plasmid was sequenced on both strands.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. Digested pJK050 and genomic DNA were ligated with T4 DNA ligase (New England Biolabs) and cosmid constructs were packaged into lambda phage with a MaxPlax Lambda Packaging Extract Kit (Epicentre) according to the manufacturer’s protocol, followed by transfection of E. coli WM4489.

    Article Title: Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance
    Article Snippet: Therefore pHXB2AF was digested with NcoI (Roche Diagnostics, Almere, The Netherlands) and NheI (New England Biolabs, Ipswich, MA, USA) to remove a 1586 bp fragment containing the NdeI site. .. PCR, using VentR ® DNA polymerase (New England BioLabs) was performed on pHXB2AF with primers, NcoI-out (5′ CAC TAG AGC TTT TAG AGG AGC TTA AGA-3′; 5614–5640), NheI-out (5′-TTT TAT TAT TTC CAA ATT GTT CTC TTA-3′; 7296–7270) and NdeI-KO (5′-TCA G AT GCT AAA GCG TAT GAT ACA G-3′; 6390–6414).

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: In brief, a region of 450 bp of human SETX cDNA was PCR amplified from the 3′ end of senataxin using Ab-1F/Ab-1R primer pair. .. This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain.

    Article Title: A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease
    Article Snippet: The tet-responsive Pol II promoter Ptight was amplified from the plasmid pTRE-Tight (Clontech Laboratories, Inc.), cloned in pGEM-T (Promega) and subcloned in pBudCE4.1 (Invitrogen) with the added restriction sites BbsI and NheI (NEB). .. The two promoters inserted in the resulting pDUAL-JU vector were sequenced in both directions to assure that the correct sequences were cloned.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Transformants were selected using the chromogenic substrate X-gal and by colony PCR. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit. .. The pET28a-MTAP construct was synthesized by treatment with T4 DNA ligase (New England Biolabs) using pre-digested pET28a vector with the same enzymes.

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways
    Article Snippet: The template clone of STAT3 (BC014482) was obtained from transOMIC technologies (Huntsville, AL, USA), and amplified the template using the following primer: forward, 5′-TGCTAGC GGACCCCTGATTTTAGCA-3′; reverse, 5′-GCTCGA GGGAACCACAAAGTTAGTAGTTT-3′. .. The PCR product was digested by NheI and XhoI restriction enzymes (NEB), and then the product was ligated into the same sites of pcDNA3.1 ( − ) expression vector. .. The MCF-7 cells were transfected with the pcDNA3.1-STAT3 plasmid and pcDNA3.1 control vector by using TransIT® -2020 transfection reagent, according to the manufacturer's instructions (Mirus Bio, Inc., Madison, WI, USA).

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: The full length PSMB5 from CMY (WT) and CMY-BR200 underwent PCR purification clean up (Qiagen). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The sequence of a ∼550-bp Xenopus rhodopsin promoter, characterized previously, was amplified by PCR to include NdeI, PmeI, and I-SceI sites at the 5′ end and an NheI site at the 3′ end. pXOP-SCFP3A-N1 and pXOP-SYFP2-N1 were generated using pSCFP3A-N1 and pSYFP2-N1. .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence. .. The SV40 early polyadenylation signal sequence present in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with NotI and AflII (New England Biolabs, Ipswich, Massachusetts) and replaced by the SV40 late polyadenylation signal sequence.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: TRPV5N358Q was obtained by in vitro mutagenesis of TRPV5-pCINeo/IRES-GFP cDNA according to the manufacturer's instructions (Stratagene, La Jolla, CA). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites. .. All constructs were verified by DNA sequence analysis.

    Article Title: A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)
    Article Snippet: In this investigation, the published sequences of human MPDU1 at the genomic level (accession number ) and of the coding region (accession number XM_008236) have been used and confirmed. .. The cDNA generated by the PCR reaction described above using SL35-F-(13F) and SL35-R-(13R) was digested with NheI (New England BioLabs Inc.) and cloned into the expression vector pTre2hyg (CLONTECH Laboratories Inc., Palo Alto, California, USA) that had been digested with NheI and EcoRV (New England BioLabs Inc.). .. After amplification in bacteria (TOP10F′; Invitrogen Corp., San Diego, California, USA), the vector constructs were purified, and the sequence of the coding region was verified by direct sequencing using a LI-COR sequencer (LI-COR Biosciences, Bad Homburg, Germany).

    Article Title: Transient AID expression for in situ mutagenesis with improved cellular fitness
    Article Snippet: PCR products were cloned by Zero Blunt PCR cloning kit (Thermo Fisher Scientific) and sequenced using standard M13 forward and reverse primers. .. Wild type AID and mutants AID genes were cloned into a lentiviral vector pAS3w.Ppuro by the HpaI and NheI restriction sites (New England BioLabs, Ipswich, MA, USA).

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The synthetic CFA MEFA gene was amplified in a PCR with Pfu Taq polymerase (Strategene, La Jolla, CA) and primers pETCFA-F (5′-GTGAGT GCTAGC GCAGTAGAGGATTTTTTCATT-3′ [NheI site underlined]) and pETCFA-R (5′-CTCT CGGCCG TTATCAGGCTCCCAAAGTCATTACAAG-3′ [EagI site underlined]). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ). .. The cloned CFA MEFA chimeric gene was verified first with DNA sequencing.

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Error prone PCR (epPCR) was performed using the GeneMorph II Random Mutagenesis kit (Stratagene, Inc.) using 25 replication cycles and 10 ng/μL template plasmid. .. The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs).

    Sonication:

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. Expression of b12 was carried out in Escherichia coli XL1 Blue competent cells (Stratagene; San Diego, CA) as previously described .

    Binding Assay:

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: A 3.7 kb segment containing the derived T allele of rs11868112 and the putative RAR binding site (see ) was amplified by nested PCR. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA).

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: In brief, a region of 450 bp of human SETX cDNA was PCR amplified from the 3′ end of senataxin using Ab-1F/Ab-1R primer pair. .. This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. A second region of 1.1 kb of SETX was PCR amplified from the 5′ end of senataxin using Ab-2F/Ab-2R primer pair.

    DNA Extraction:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Nucleic Acid Electrophoresis:

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: The DNA solutions were analyzed by field inversion gel electrophoresis on a 1% agarose/TBE gel and the fraction containing ~20–60 kb DNA fragments was treated with shrimp alkaline phosphatase (Roche Diagnostics). .. Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The synthetic CFA MEFA gene was amplified in a PCR with Pfu Taq polymerase (Strategene, La Jolla, CA) and primers pETCFA-F (5′-GTGAGT GCTAGC GCAGTAGAGGATTTTTTCATT-3′ [NheI site underlined]) and pETCFA-R (5′-CTCT CGGCCG TTATCAGGCTCCCAAAGTCATTACAAG-3′ [EagI site underlined]). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ). .. The cloned CFA MEFA chimeric gene was verified first with DNA sequencing.

    Magnetic Beads:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA). .. The linker-ligated DNA was amplified using SNX as a linker-specific primer for polymerase chain reaction (PCR).

    Mutagenesis:

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: Paragraph title: Production of the CL309/310CH mutant ... PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: TRPV5N358Q was obtained by in vitro mutagenesis of TRPV5-pCINeo/IRES-GFP cDNA according to the manufacturer's instructions (Stratagene, La Jolla, CA). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites.

    Article Title: Transient AID expression for in situ mutagenesis with improved cellular fitness
    Article Snippet: Paragraph title: AID mutant construction ... Wild type AID and mutants AID genes were cloned into a lentiviral vector pAS3w.Ppuro by the HpaI and NheI restriction sites (New England BioLabs, Ipswich, MA, USA).

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: This CFA MEFA chimeric gene, after silent mutation to fit PCR primer design and cloning purposes, was synthesized (Integrated DNA Technologies, Inc., Coralville, IA). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Paragraph title: Mutant Library Generation ... The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs).

    Isolation:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Genomic DNA was isolated from one individual using the TNES-urea buffer method [ ]. .. Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA).

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: To clone the PSMB5 subunit, RNA was isolated from CMY-BR200 and from the parental CMY cell lines using RNeasy mini kit (Qiagen, Gaithersburg, MD). cDNA was prepared by reverse transcription using iScript™ (Bio-Rad) and whole length PSMB5 was amplified by PCR using the following primer set: Forward: 5′-ATTA GCTAGC AGACATGGCGCTTGCCAGCGTGTT-3′ containing the NheI restriction site (GCTAGC), and Reverse: 5′-TATA CTCGAG TCAGGGGGTAGAGCCACTATACTTCT-3′ containing the XhoI restriction site (CTCGAG) (LifeTechnologies). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Subcloning:

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Forward (5’-CTGGCTAGC ATGTCTAAAGTTAAGGTTGGAATTATTG-3’) and reverse (5’- CTGCTCGAG CCAATTTACTTCATGTTTATTTGTCATTAC-3’) primers containing NheI and XhoI restriction sites (in italics) were designed for subcloning into the pET28a expression vector. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Labeling:

    Article Title:
    Article Snippet: NheI, BstApI, ApaI, BsaWI, BbsI, and HindIII were obtained from New England Biolabs (Ipswich, MA). .. NheI, BstApI, ApaI, BsaWI, BbsI, and HindIII were obtained from New England Biolabs (Ipswich, MA).

    Purification:

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. To verify incorporation of the desired mutation and to check for PCR-induced errors, the entire coding sequence of the new expression plasmid was sequenced on both strands.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: The DNA solutions were analyzed by field inversion gel electrophoresis on a 1% agarose/TBE gel and the fraction containing ~20–60 kb DNA fragments was treated with shrimp alkaline phosphatase (Roche Diagnostics). .. Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. The cosmid was further treated with Bam HI (Invitrogen).

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant Sm MTAP ... The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: The full length PSMB5 from CMY (WT) and CMY-BR200 underwent PCR purification clean up (Qiagen). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. To purify b12, bacterial cell pellets were resuspended in PBS pH 7.4 containing a final concentration of 0.2 mM PMSF (#P-470-10; Gold Biotechnology, Inc.; St. Louis, MO) and sonicated to produce cellular lysate.

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Error prone PCR (epPCR) was performed using the GeneMorph II Random Mutagenesis kit (Stratagene, Inc.) using 25 replication cycles and 10 ng/μL template plasmid. .. The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs). .. Ligations were performed for 15 hours at 4 °C using T4 DNA ligase (Promega, Inc.) and contained 3:1 insert:vector ratio.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: From the total mRNA from the adult worm, strand cDNAs were synthesized by RT-PCR employing the SuperScript III First-Strand Synthesis System from Promega. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    shRNA:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: Knockdown of Klf15 was achieved using a Klf15 shRNA construct (shKlf15 ) or empty control virus shRNA (Ctl ) as previously described . .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Article Title: Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells
    Article Snippet: Following plasmids were obtained from Sigma–Aldrich and their catalogue number is given in brackets: TCF4 shRNA (TRCN0000012094), Ctbp1 shRNA (TRCN0000085774), TCF4 shRNA targeting 3′ UTR (TRCN0000012093), β-catenin shRNA targeting 3′ UTR (TRCN0000012688), Ctbp1 shRNA targeting 3′ UTR (TRCN0000085773). .. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs.

    IA:

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: This CFA MEFA chimeric gene, after silent mutation to fit PCR primer design and cloning purposes, was synthesized (Integrated DNA Technologies, Inc., Coralville, IA). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ).

    In Situ Hybridization:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA).

    Plasmid Preparation:

    Article Title: Novel Microsatellite Markers of Meretrix petechialis and Cross-species Amplification in Related Taxa (Bivalvia: Veneroida)
    Article Snippet: Extracted DNA was digested with a restriction enzyme mixture containing Alu I, Rsa I, Nhe I and Hha I (New England Biolabs, Beverly, MA, USA). .. For enrichment, the DNA was denatured, hybridized to biotin-labeled repeat sequence (GT)10 probes and then attached to streptavidin-coated magnetic beads (Promega, Madison, WI, USA).

    Article Title: Allele-Specific Down-Regulation of RPTOR Expression Induced by Retinoids Contributes to Climate Adaptations
    Article Snippet: For both PCR reactions, iProof High-Fidelity DNA Polymerase (Bio-Rad) was used to avoid the introduction of mutations. .. After digestion with Nhe I and Xho I (New England Biolabs, Ipswich, MA), the DNA fragment was cloned into the pGL3-promoter vector (Promega, Hercules, CA). .. The plasmid with the ancestral allele (C) was generated with the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) using primer pair 5′-GCCCTTGACAAGCT C ACAAACTTGTAGGAGGG-3′ and 5′-CCCTCCTACAAGTTTGT G AGCTTGTCAAGGGC-3′ (target in bold) according to the manufacturer's recommendations.

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: To clone the Fgf15 promoter reporter construct, mouse genomic DNA was first isolated from mouse liver using a genomic DNA isolation kit (Qiagen, Germantown, MD, USA). .. The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. The sequences of primers for Fgf15 promoter: forward, 5′-gatcGCTAGCGCTGTCTTCAGACACAC-3′ and reverse, 5′-GCTCGGGCCACCGCACCTCGAGgatc-3′.

    Article Title: Developing the IVIG biomimetic, Hexa-Fc, for drug and vaccine applications
    Article Snippet: The N297A mutant was similarly constructed from the same vector using the internal mismatched primer mut-3:5′-CTCGTCATGCGGTCGTGCATG -3′ and its complement to incorporate an AAC to GCC substitution and Fcmut-1:5′-ACCCTGCTTGCTCAACTCT-3′ and Fcmut-1:3′-TTGATGAGTTTGGACAAACCA-5′ as flanking primers. .. PCR products were then digested using Bgl II and Nhe I (New England Biolabs) and cloned back into the wild-type vector to generate pFUSE-hIgG1-Fc-TP-CL309/310CH or N297A. .. To verify incorporation of the desired mutation and to check for PCR-induced errors, the entire coding sequence of the new expression plasmid was sequenced on both strands.

    Article Title: Biosynthesis of the Antimicrobial Peptide Epilancin 15X and Its N-terminal Lactate
    Article Snippet: Cosmid pJK050 was digested with Nhe I (New England Biolabs), treated with shrimp alkaline phosphatase (Roche Diagnostics), and purified. .. Library clones were screened by PCR for the elxA gene using forward primer elxA-F1 and reverse primer elxA-R2, or for the elxC gene with forward primer elxC-596F and reverse primer elxC-781R, Taq polymerase (Invitrogen), and 1x PCR premix A (Epicentre).

    Article Title: Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage
    Article Snippet: In brief, a region of 450 bp of human SETX cDNA was PCR amplified from the 3′ end of senataxin using Ab-1F/Ab-1R primer pair. .. This SETX fragment was subsequently cloned into NheI and NotI sites of pTYB1 plasmid (New England Biolabs, Inc.), containing a C-terminal chitin binding domain. .. A second region of 1.1 kb of SETX was PCR amplified from the 5′ end of senataxin using Ab-2F/Ab-2R primer pair.

    Article Title: A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease
    Article Snippet: SOFA-HDV-Rz cleavage assays were performed in the presence of 1 pmol of either 3′- (NS substrate) or internally 32 P-labelled target mRNA (all other substrates) mixed with SOFA-HDV-Rz (20 pmol) in a 20 µL mixture containing 50 mM Tris-HCl, pH 7.5, and 10 mM MgCl2 and then incubated at 37°C for 2 h. The reactions were stopped by the addition of denaturing loading buffer, separated on denaturing 5% PAGE gels, and then analyzed by autoradiography on a Phosphorscreen (GE Healthcare). .. The tet-responsive Pol II promoter Ptight was amplified from the plasmid pTRE-Tight (Clontech Laboratories, Inc.), cloned in pGEM-T (Promega) and subcloned in pBudCE4.1 (Invitrogen) with the added restriction sites BbsI and NheI (NEB). .. The Pol III promoter PhHI was amplified from the plasmid psiRNA-hH1GFPzeo (InvivoGen) and cloned in the engineered plasmid with the added restriction sites BspHI and SbfI.

    Article Title: Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells
    Article Snippet: Following plasmids were generous gifts from Prof. Sanjeev Galande (IISER Pune, India): RFP tagged constructs of full length β-catenin (β-cat FL-RFP), N terminal β-catenin (β-cat N-RFP), C- terminal β-catenin (β-cat C-RFP), GST tagged full-length β-catenin (β-cat FL-GST), Flag tagged TCF4 construct (Flag-TCF4) and Flag tagged Ctbp1 plasmid construct (Flag-Ctbp1) were kind gifts from Prof. G. Chinnadurai (Saint Louis University, School of Medicine). .. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs.

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: The RT product was used as a template for PCR; after adenylation, the amplification product was cloned into the pTZ57R/T vector and transformed into Escherichia coli DH5α cells. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Docosahexaenoic acid inhibits 12-O-tetradecanoylphorbol-13- acetate-induced fascin-1-dependent breast cancer cell migration by suppressing the PKCδ- and Wnt-1/β-catenin-mediated pathways
    Article Snippet: The template clone of STAT3 (BC014482) was obtained from transOMIC technologies (Huntsville, AL, USA), and amplified the template using the following primer: forward, 5′-TGCTAGC GGACCCCTGATTTTAGCA-3′; reverse, 5′-GCTCGA GGGAACCACAAAGTTAGTAGTTT-3′. .. The PCR product was digested by NheI and XhoI restriction enzymes (NEB), and then the product was ligated into the same sites of pcDNA3.1 ( − ) expression vector. .. The MCF-7 cells were transfected with the pcDNA3.1-STAT3 plasmid and pcDNA3.1 control vector by using TransIT® -2020 transfection reagent, according to the manufacturer's instructions (Mirus Bio, Inc., Madison, WI, USA).

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: The full length PSMB5 from CMY (WT) and CMY-BR200 underwent PCR purification clean up (Qiagen). .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. Digested products were separated on an agarose gel, extracted and purified (Qiagen).

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The anti-Env b12 Fab fragment recombinant expression vector was a kind gift from Dr. Dennis Burton . .. The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. Expression of b12 was carried out in Escherichia coli XL1 Blue competent cells (Stratagene; San Diego, CA) as previously described .

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence. .. The SV40 early polyadenylation signal sequence present in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with NotI and AflII (New England Biolabs, Ipswich, Massachusetts) and replaced by the SV40 late polyadenylation signal sequence.

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: The pCINeo/IRES-GFP plasmid encoding TRPV5 was generated as described previously ( ). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites.

    Article Title: A mutation in the human MPDU1 gene causes congenital disorder of glycosylation type If (CDG-If)
    Article Snippet: In this investigation, the published sequences of human MPDU1 at the genomic level (accession number ) and of the coding region (accession number XM_008236) have been used and confirmed. .. The cDNA generated by the PCR reaction described above using SL35-F-(13F) and SL35-R-(13R) was digested with NheI (New England BioLabs Inc.) and cloned into the expression vector pTre2hyg (CLONTECH Laboratories Inc., Palo Alto, California, USA) that had been digested with NheI and EcoRV (New England BioLabs Inc.). .. After amplification in bacteria (TOP10F′; Invitrogen Corp., San Diego, California, USA), the vector constructs were purified, and the sequence of the coding region was verified by direct sequencing using a LI-COR sequencer (LI-COR Biosciences, Bad Homburg, Germany).

    Article Title: Transient AID expression for in situ mutagenesis with improved cellular fitness
    Article Snippet: Site directed mutagenesis using QuikChange™ Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) was used to generate the AID S38A mutant with compromised activity and AID-M6A with total loss of activity . .. Wild type AID and mutants AID genes were cloned into a lentiviral vector pAS3w.Ppuro by the HpaI and NheI restriction sites (New England BioLabs, Ipswich, MA, USA). .. An HA tag epitope (YPYDVPDYA) was fused to the C-terminus of AID and AID mutants to allow detection by immunoblot analysis.

    Article Title: Multiepitope Fusion Antigen Induces Broadly Protective Antibodies That Prevent Adherence of Escherichia coli Strains Expressing Colonization Factor Antigen I (CFA/I), CFA/II, and CFA/IV
    Article Snippet: The synthetic CFA MEFA gene was amplified in a PCR with Pfu Taq polymerase (Strategene, La Jolla, CA) and primers pETCFA-F (5′-GTGAGT GCTAGC GCAGTAGAGGATTTTTTCATT-3′ [NheI site underlined]) and pETCFA-R (5′-CTCT CGGCCG TTATCAGGCTCCCAAAGTCATTACAAG-3′ [EagI site underlined]). .. PCR products were extracted by gel electrophoresis, digested with NheI/EagI restriction enzymes (New England BioLabs, Ipswich, MA), and ligated into expression vector pET28α using standard protocols ( ). .. The cloned CFA MEFA chimeric gene was verified first with DNA sequencing.

    Article Title: Bioretrosynthetic construction of a didanosine biosynthetic pathway
    Article Snippet: Error prone PCR (epPCR) was performed using the GeneMorph II Random Mutagenesis kit (Stratagene, Inc.) using 25 replication cycles and 10 ng/μL template plasmid. .. The epPCR products were gel purified, digested with NheI /XhoI (New England Biolabs, Inc.) and gel purified before ligation into purified pET28a+ vector that had been similarly digested and treated with alkaline phosphatase (New England Biolabs). .. Ligations were performed for 15 hours at 4 °C using T4 DNA ligase (Promega, Inc.) and contained 3:1 insert:vector ratio.

    RNA Extraction:

    Article Title: Circadian control of bile acid synthesis by a KLF15-Fgf15 axis
    Article Snippet: The mouse Fgf15 promoter from −2,952 to −60 (2,893 bp) was amplified from genomic DNA by PCR (CloneAmp HiFi PCR Premix, Clontech, Mountain View, CA, USA) and inserted into a pGL3 luciferase reporter vector using restriction sites digested by Nhe I and Xho I (New England Biolabs Inc., Ipswich, MA, USA). .. ISH of Fgf15 expression in the mouse ileum was carried out using a truncated clone of Fgf15 cDNA as an ISH probe, which has been previously described .

    Selection:

    Article Title: Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells
    Article Snippet: PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning. .. PCR products, and pcDNA 3.1 Hygro (+) plasmid vector (LifeTechnologies) were then each treated with NheI and XhoI restriction enzymes (NEB, Ipswich, MA, USA) for preparation for cloning.

    Agarose Gel Electrophoresis:

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Transformants were selected using the chromogenic substrate X-gal and by colony PCR. .. The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit. .. The pET28a-MTAP construct was synthesized by treatment with T4 DNA ligase (New England Biolabs) using pre-digested pET28a vector with the same enzymes.

    In Vitro:

    Article Title: The Epithelial Calcium Channel TRPV5 Is Regulated Differentially by Klotho and Sialidase
    Article Snippet: TRPV5N358Q was obtained by in vitro mutagenesis of TRPV5-pCINeo/IRES-GFP cDNA according to the manufacturer's instructions (Stratagene, La Jolla, CA). .. The pcDendra-2 (Evrogen, Moscow, Russia) was amplified by PCR and ligated into the pCINeo/IRES-HA TRPV5 construct using the NheI and EcoNI (New England Biolabs, Ipswich, MA) restriction sites.

    Transgenic Assay:

    Article Title: F?rster resonance energy transfer as a tool to study photoreceptor biology
    Article Snippet: The vectors pXOP-SCFP3A-N1, pXOP-SYFP2-N1, and pXOP-SYFP2-SCFP3A were constructed for use in the generation of transgenic Xenopus laevis . .. The CMV promoter sequence in pSCFP3A-N1 and pSYFP2-N1 was removed by digestion with the restriction enzymes NdeI and NheI (New England Biolabs, Ipswich, Massachusetts) and was replaced by the PCR-amplified Xenopus rhodopsin promoter sequence.

    Affinity Chromatography:

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. To purify b12, bacterial cell pellets were resuspended in PBS pH 7.4 containing a final concentration of 0.2 mM PMSF (#P-470-10; Gold Biotechnology, Inc.; St. Louis, MO) and sonicated to produce cellular lysate.

    Concentration Assay:

    Article Title: Mechanism of Wnt signaling induced down regulation of mrhl long non-coding RNA in mouse spermatogonial cells
    Article Snippet: Plasmids were used for transient transfection in Gc1-Spg cells at a final concentration of 1.5 μg/ml using Lipofectamine 2000 as per manufacturer's instructions. .. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs.

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone. .. Expression of b12 was carried out in Escherichia coli XL1 Blue competent cells (Stratagene; San Diego, CA) as previously described .

    Recombinant:

    Article Title: Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway
    Article Snippet: Paragraph title: Cloning, expression and purification of recombinant Sm MTAP ... The MTAP gene was digested with NheI and XhoI (New England Biolabs) and recovered on a 1% agarose gel using the Promega Wizard SV Gel and PCR Clean Up kit.

    Article Title: Single molecule fate of HIV-1 envelope reveals late-stage viral lattice incorporation
    Article Snippet: Paragraph title: Recombinant antibody fragment production ... The pCOMB3H-b12 expression vector was deleted for the pIII gene by digestion with SpeI and NheI (New England Biolabs; Ipswich, MA) and ligation of the vector backbone.

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    New England Biolabs kpni
    <t>PFGE-based</t> clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and <t>KpnI,</t> and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
    Kpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kpni/product/New England Biolabs
    Average 99 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs kpni hf
    <t>PFGE-based</t> clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and <t>KpnI,</t> and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A
    Kpni Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Journal:

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE-based clustering of C. coli strains investigated in this study. All 68 strains were analyzed by PFGE with SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Two strains (1004 A

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Journal:

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group I strains. Nineteen MDR strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. The line labeled “a” indicates

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques: Labeling

    PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Journal:

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of group II strains. Forty-two strains were characterized by PFGE using SmaI and KpnI, and cluster analysis of the patterns was performed by BioNumerics as described in Materials and Methods. Strains which were not resistant to all six antibiotics

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Journal:

    Article Title: Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

    doi: 10.1128/AEM.02346-06

    Figure Lengend Snippet: PFGE profiles of 13 strains with various STs but a conserved PFGE profile (type a ). Cluster analysis of the SmaI and KpnI patterns was performed by BioNumerics as described in Materials and Methods.

    Article Snippet: PFGE was performed using SmaI and KpnI (New England Biolabs) by following the protocol described by Ribot et al. ( ) with a few minor modifications; specifically, the prerestriction step was eliminated and the gels were electrophoresed for 22 h. TIFF images of banding patterns resulting from PFGE and fla typing were analyzed by BioNumerics (version 4.6; Applied Maths).

    Techniques:

    Molecular analyses of L. incisa clones transformed with a pRbcS450 construct. (A) PCR analysis: gDNA was amplified with BleF and BleR primers yielding a 415-bp fragment. Lanes: (M) DNA ladder; (-) no template, (Ble) ble plasmid control, (wt) negative control (non-transformed cells); five transformed clones. (B) Southern blot analysis: gDNA isolated from both transgenic and non-transgenic cells, as well as plasmid DNA (pRbcS450), were digested with KpnI restriction enzyme. The blot was hybridized with a probe derived from a 415-bp amplified fragment of the ble gene. Lanes: (M) 1 Kb ladder; (plasmid) positive control; five transformed clones; (wt) negative control (non-transformed cells).

    Journal: PLoS ONE

    Article Title: Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

    doi: 10.1371/journal.pone.0105223

    Figure Lengend Snippet: Molecular analyses of L. incisa clones transformed with a pRbcS450 construct. (A) PCR analysis: gDNA was amplified with BleF and BleR primers yielding a 415-bp fragment. Lanes: (M) DNA ladder; (-) no template, (Ble) ble plasmid control, (wt) negative control (non-transformed cells); five transformed clones. (B) Southern blot analysis: gDNA isolated from both transgenic and non-transgenic cells, as well as plasmid DNA (pRbcS450), were digested with KpnI restriction enzyme. The blot was hybridized with a probe derived from a 415-bp amplified fragment of the ble gene. Lanes: (M) 1 Kb ladder; (plasmid) positive control; five transformed clones; (wt) negative control (non-transformed cells).

    Article Snippet: Briefly, L. incisa genomic DNA (5 µg) was digested with KpnI endonuclease (NEB), desalinated by EtOH precipitation and separated by electrophoresis on a 0.7% agarose (Seakem, Lonza) gel.

    Techniques: Clone Assay, Transformation Assay, Construct, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Negative Control, Southern Blot, Isolation, Transgenic Assay, Derivative Assay, Positive Control

    Schematics of the LiRbcS transformation constructs and corresponding average number of resultant colonies per plate. The number of colonies is the average of 12 different transformation events. The blue boxes represent the ble coding sequence, and the green boxes represent the LiRbcS promoter and terminating sequence. KpnI restriction site used in Southern blot hybridization is marked on the construct that was used for L. incisa transformation.

    Journal: PLoS ONE

    Article Title: Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

    doi: 10.1371/journal.pone.0105223

    Figure Lengend Snippet: Schematics of the LiRbcS transformation constructs and corresponding average number of resultant colonies per plate. The number of colonies is the average of 12 different transformation events. The blue boxes represent the ble coding sequence, and the green boxes represent the LiRbcS promoter and terminating sequence. KpnI restriction site used in Southern blot hybridization is marked on the construct that was used for L. incisa transformation.

    Article Snippet: Briefly, L. incisa genomic DNA (5 µg) was digested with KpnI endonuclease (NEB), desalinated by EtOH precipitation and separated by electrophoresis on a 0.7% agarose (Seakem, Lonza) gel.

    Techniques: Transformation Assay, Construct, Sequencing, Southern Blot, Hybridization

    Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Journal: Tropical Medicine and Infectious Disease

    Article Title: Pathogenicity and Immunogenicity of Recombinant Rabies Viruses Expressing the Lagos Bat Virus Matrix and Glycoprotein: Perspectives for a Pan-Lyssavirus Vaccine

    doi: 10.3390/tropicalmed2030037

    Figure Lengend Snippet: Schematic representation of the construction of recombinant viruses. Restriction enzyme sites are indicated ( XmaI , PacI , AvrII , KpnI . BsiWI , AsiSI , NheI and AscI ). GAS represents the SPBN G gene with two amino acid substitutions (Asn 194 to Ser and Arg 333 to Glu). The following abbreviations were used: N, nucleoprotein; M, matrix protein; G, glycoprotein; L, RNA-dependent RNA polymerase. LBVM and LBVG represent the LBV M and G gene respectively.

    Article Snippet: To replace both the M and G genes of SPBN with those of the LBVAFR1999, the KpnI site was introduced upstream of the pSPBN-LBVG M gene start signal, through digestion of another pSPBN (that contains the KpnI site) with AvrII and KpnI (New England Biolabs, Ipswich, MA, USA) ( ).

    Techniques: Recombinant