nhei hf  (New England Biolabs)


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    Name:
    NheI HF
    Description:
    NheI HF 5 000 units
    Catalog Number:
    r3131l
    Price:
    282
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs nhei hf
    NheI HF
    NheI HF 5 000 units
    https://www.bioz.com/result/nhei hf/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    nhei hf - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: Paragraph title: Cloning of Antibody Expression Vector. ... The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction.

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were contransformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified before experiments.

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: .. Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: Homology arms were inserted into pUC19 using an In-Fusion HD Cloning Kit. .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: The resulting fragment was cloned into pDONR221 and from there transferred into pTGW through Gateway recombination. .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.).

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were co-transformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified prior to experiments.

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: Two series of random mutagenesis and FACS-based selections (named I and II) were applied to improve both the binding affinity and crossreactivity of three promiscuous clones: CK1, CK2, and CK4. .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Amplification:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: The DNA fragments encoding the heavy chains and light chains of Syn and BVK, along with the linkers (coiled-coil or β-strand) ( , ) were also synthesized by IDT and amplified by PCR. .. The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction.

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: The fragment before and containing the H2 was amplified with the pCTFwd Recomb and appropriate PreH2 primers, and the fragment after and also containing the H2 was amplified using the pcTRev Recomb and proper PostH2 primers (Primer sequences in Supplementary Table 1). .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs).

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: .. Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: Tyr homology arms were amplified by PCR using the primers listed in Table (5′-homology arm and 3′-homology arm) using the template as above. .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.). .. In this vector we finally inserted through restriction and ligation the rest of the Tango1 coding sequence, amplified from pDONR221-Tango1 with primers SpeITango1-F: 5′-GGACTAGTGCGACTCTCTCCGACAAGCG-3′ and XhoITango1-R: 5′-CCGCTCGAGTACCTCGCTGTAGGGTCGCG-3′.

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: For each motif, all positional variants of the H3-containing fragment were amplified with the pCTCON2 RecombFwd and appropriate PreH3 primers, and the fragment after and also containing the H3 was amplified using the pCTCON2 RecombRev and proper PostH3 primers (Primer sequences in ). .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs).

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: To ensure a mutagenesis rate of approximately 1–2 amino-acid mutated residues distributed randomly throughout the entire gene, 1 ng of DNA template encoding the CK1, CK2, and CK4 binders were PCR amplified for 15 cycles using Taq DNA polymerase (New England BioLabs), analog nucleotides (2 μM 8-oxo-dGTP and 2 μM dPTP) and flanking oligonucleotide primers (forward: 5′-GGAGGCGGTAGCGGAGGCGGAGGGTCGGCTAGC-3′; reverse: 5′-GTCCTCTTCAGAAATAAGCTTTTGTTCGGAT-3′; Integrated DNA Technologies). .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Synthesized:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: The DNA fragments encoding the heavy chains and light chains of Syn and BVK, along with the linkers (coiled-coil or β-strand) ( , ) were also synthesized by IDT and amplified by PCR. .. The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction.

    Neutralization:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. The gel was subsequently incubated in Denaturing solution (1.5 M NaCl, 0.5M NaOH) for 45 minutes; Depurination solution (0.2N HCl) for 15 minutes; Neutralization Solution (1M Tris pH7.4, 1.5M NaCl) twice for 30 minutes; and then transferred onto a membrane (Amersham Hybond-N RPN303N) using 10% SSC buffer at room temperature for overnight.

    TA Cloning:

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. Splicing was analysed by RT-PCR (OneStep RT-PCR kit; Qiagen) using primers 5′-CACCTTCCTGGGTCTGCCTCAGTACC-3′ and 5′-GGCGCCACCAGCAGGACCAGCA-3′ according to manufacturer’s protocol. cDNA amplification products were cloned into vectors (pCR® II-TOPO® using TOPO TA cloning technique, Invitrogen) according to manufacturers protocol for subsequent sequencing.

    Construct:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction. .. The final expression vectors for the fusion antibodies were constructed by in-frame ligation of the assembled DNA into the pFuse backbone (Invivogen) using T4 DNA ligase.

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: Designed overlap sequences were located at the two ends of the scFv construct as well as directly in the H2 region. .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs).

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Paragraph title: Established constructs ... Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation.

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: .. In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA). .. The reporter constructs were then used to transform competent Escherichia coli DH5α cells (New England Biolabs) and plated on 100 μg/mL ampicillin Luria-Bertani (LB) agar plates.

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: Targeting vector construction For Tyr -repair of albino rats, a targeting ssAAV vector plasmid, pssAAV_rTyr -repair, was constructed. .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: Designed overlap sequences were located at the two ends of the scFv construct as well as directly in the H3 region. .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs).

    Article Title: The Yin and Yang of SagS: Distinct Residues in the HmsP Domain of SagS Independently Regulate Biofilm Formation and Biofilm Drug Tolerance
    Article Snippet: Furthermore, alanine cassette mutants were constructed in regions that contained two or more conserved residues. .. Site-directed mutated variants of sagS were subsequently subcloned into the pMJT-1 vector using the oligonucleotides listed in in the supplemental material and the restriction enzymes NheI-HF and SacI-HF (New England Biolabs).

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Pre-mixed DNA linearized vector and PCR insert (1 μg μL−1 ) was electroporated into freshly prepare EBY100 competent cells, where the full constructs are reassembled via homologous recombination .

    Incubation:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. The gel was subsequently incubated in Denaturing solution (1.5 M NaCl, 0.5M NaOH) for 45 minutes; Depurination solution (0.2N HCl) for 15 minutes; Neutralization Solution (1M Tris pH7.4, 1.5M NaCl) twice for 30 minutes; and then transferred onto a membrane (Amersham Hybond-N RPN303N) using 10% SSC buffer at room temperature for overnight.

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Surviving colonies were added to 4 mL liquid LB/kanamycin (50 mg/L) and incubated in shake flasks at 37 °C, 250 rpm for 10–16 hours.

    Article Title: A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture
    Article Snippet: Digestions were performed overnight at 37°C with 400U NheI-HF (NEB) for the IPT3 locus, 400U BglII (NEB) for the IPT7 locus or 400U MfeI (NEB) for the KRP7 locus. .. Restriction enzymes were inactivated by addition of 1,6% SDS and incubation at 65°C for 20min.

    Luciferase:

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: Paragraph title: ABCB1 promoter haplotype luciferase reporter construct generation ... In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA).

    Activity Assay:

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: For the current study, four haplotypes were evaluated , namely, the ancestral haplotype 1 representing the reference promoter activity (100%), haplotypes 4 and 29 with significantly higher basal promoter activity than haplotype 1 (390% and 350%, respectively), and haplotype 30 with significantly lower basal promoter activity than haplotype 1 (6%). .. In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA).

    Expressing:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: Paragraph title: Cloning of Antibody Expression Vector. ... The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction.

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: .. Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Saturated cultures were added to 100 mL of LB, incubated 37 °C, 250 rpm until reaching an optical density between 0.5 and 1.0 upon which 0.1 mL 0.5 mM isopropyl b-D-1-thiogalactopyranoside was added to induce protein expression.

    Modification:

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: UAS-SP.GFP.Tango1 To express Tango1 N-terminally tagged with GFP after its signal peptide, we modified pTGW (UAST N-terminal GFP, Drosophila Carnegie Vector collection) by adding the signal peptide of Tango1 5′ to the GFP sequence as well as SpeI and XhoI restriction sites 3′ to the GFP sequence. .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.).

    Transformation Assay:

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were contransformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified before experiments.

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: .. Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were co-transformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified prior to experiments.

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Transformed cultures were recovered and expanded in SD-SCAA.

    Hybridization:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. The membrane was crosslinked by UV, and incubated in pre-heated hybridization buffer (Roche 11796895001) for 30 minutes at 42°C.

    Transfection:

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Southern Blot:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: Paragraph title: Southern blot ... 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight.

    Ligation:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction. .. The final expression vectors for the fusion antibodies were constructed by in-frame ligation of the assembled DNA into the pFuse backbone (Invivogen) using T4 DNA ligase.

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.). .. The excised GFP sequence was replaced through restriction and ligation with T4 DNA ligase (cat#M0202L; New England Biolabs, Inc.) by the signal peptide of Tango1 (26 first amino acids, predicted by SignalP 4.1), which we PCR-amplified from pDONR221-Tango1 using primers that added the appropriate restriction sites (XbaISP-F: 5′-GGCTCTAGAATGCGGCTGACCAACGAGA-3′ and NheISP-R: 5′-CTAGCTAGCAGCCCACGTCAAAGTTGGAA-3′).

    Generated:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. Dig-labeled Rab27 ORF probe was generated by PCR using the following primers: 5′-TTGACGTTGGCGCCGGTGCA-3′, 5′-TGAGCCTCTGCAATTAGCCGGAT-3′, labeled with Dig using Klenow (NEB) with labeling mix (NEB), boiled for 5 minutes to denature, and added to the membrane for hybridization overnight at 45°C.

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: .. In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA). .. The reporter constructs were then used to transform competent Escherichia coli DH5α cells (New England Biolabs) and plated on 100 μg/mL ampicillin Luria-Bertani (LB) agar plates.

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: Random mutagenesis libraries were generated by error-prone PCR . .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Polymerase Chain Reaction:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. Dig-labeled Rab27 ORF probe was generated by PCR using the following primers: 5′-TTGACGTTGGCGCCGGTGCA-3′, 5′-TGAGCCTCTGCAATTAGCCGGAT-3′, labeled with Dig using Klenow (NEB) with labeling mix (NEB), boiled for 5 minutes to denature, and added to the membrane for hybridization overnight at 45°C.

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: .. The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction. .. The final expression vectors for the fusion antibodies were constructed by in-frame ligation of the assembled DNA into the pFuse backbone (Invivogen) using T4 DNA ligase.

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Established constructs The genomic fragments comprising AMN exon 8 to exon 12 (c.782-1327) were amplified through PCR from a healthy control subject and the index patient of family 3 (homozygous for c.1006 + 11_1008del) using the following AMN specific primers; 5′-CCCTCCCGCTAGCATGGCCGTTGTGTTGCTGACCCA-3′ containing the NheI restriction site and an in frame ATG start codon and primer 5′-ATTCCCCTCGAGTCATGACGAAGTAACTGTGGCTGGT-3′ containing the Xho I restriction site and an in frame TGA stop codon. .. Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation.

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: Tyr homology arms were amplified by PCR using the primers listed in Table (5′-homology arm and 3′-homology arm) using the template as above. .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: To do this, the GFP sequence was PCR-amplified from pTGW with primers adding att sites and restriction sites for NheI (5′) and XhoI followed by SpeI (3′) as follows: attNheIGFP-F: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCTAGCTAGCATGGTGAGCAAGGGCGAGGA-3′; attXhoISpeIGFP-R: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCGAGCGGGACTAGTCTTGTACAGCTCGTCCATGC-3′. .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.).

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Pre-mixed DNA linearized vector and PCR insert (1 μg μL−1 ) was electroporated into freshly prepare EBY100 competent cells, where the full constructs are reassembled via homologous recombination .

    DNA Sequencing:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction. .. The sequences of the resulting mammalian expression vectors were confirmed by DNA sequencing (GENEWIZ).

    Sequencing:

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were contransformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified before experiments.

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. Splicing was analysed by RT-PCR (OneStep RT-PCR kit; Qiagen) using primers 5′-CACCTTCCTGGGTCTGCCTCAGTACC-3′ and 5′-GGCGCCACCAGCAGGACCAGCA-3′ according to manufacturer’s protocol. cDNA amplification products were cloned into vectors (pCR® II-TOPO® using TOPO TA cloning technique, Invitrogen) according to manufacturers protocol for subsequent sequencing.

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2. ..

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.). .. The excised GFP sequence was replaced through restriction and ligation with T4 DNA ligase (cat#M0202L; New England Biolabs, Inc.) by the signal peptide of Tango1 (26 first amino acids, predicted by SignalP 4.1), which we PCR-amplified from pDONR221-Tango1 using primers that added the appropriate restriction sites (XbaISP-F: 5′-GGCTCTAGAATGCGGCTGACCAACGAGA-3′ and NheISP-R: 5′-CTAGCTAGCAGCCCACGTCAAAGTTGGAA-3′).

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were co-transformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified prior to experiments.

    Binding Assay:

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: Two series of random mutagenesis and FACS-based selections (named I and II) were applied to improve both the binding affinity and crossreactivity of three promiscuous clones: CK1, CK2, and CK4. .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Mutagenesis:

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: Paragraph title: Mutant construction ... The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs).

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. The c.3335G > A (p.Gly1112Glu) identified in family 5, was introduced into a previously described fragment of human cubilin, encoding CUB domains 5–8 [ ], through site-directed, ligase-independent mutagenesis (SLIM)[ ] using the following primers (Ft: 5′-CAGAGATGAAGGCTATGAAAAATCACCATTGCTGGG-3′; Rt: 5′-TCATAGCCTTCATCTCTGATTTCCAGAAAATCTGTA-3′; Fs: 5′-AAAATCACCATTGCTGGG-3′; Rs: 5′-ATTTCCAGAAAATCTGTA-3′).

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: These homology arms include the Tyr _repair cassette, which has corrected sequences containing an in-frame silent mutation to provide an Sna BI site for RFLP analysis. .. After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: The Yin and Yang of SagS: Distinct Residues in the HmsP Domain of SagS Independently Regulate Biofilm Formation and Biofilm Drug Tolerance
    Article Snippet: Conserved residues were substituted for with alanine (or serine if the conserved residue was an alanine) by means of the GeneArt site-directed mutagenesis kit (Invitrogen) and the Q5 site-directed mutagenesis kit (New England Biolabs) according to the manufacturer’s protocol. .. Site-directed mutated variants of sagS were subsequently subcloned into the pMJT-1 vector using the oligonucleotides listed in in the supplemental material and the restriction enzymes NheI-HF and SacI-HF (New England Biolabs).

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: To ensure a mutagenesis rate of approximately 1–2 amino-acid mutated residues distributed randomly throughout the entire gene, 1 ng of DNA template encoding the CK1, CK2, and CK4 binders were PCR amplified for 15 cycles using Taq DNA polymerase (New England BioLabs), analog nucleotides (2 μM 8-oxo-dGTP and 2 μM dPTP) and flanking oligonucleotide primers (forward: 5′-GGAGGCGGTAGCGGAGGCGGAGGGTCGGCTAGC-3′; reverse: 5′-GTCCTCTTCAGAAATAAGCTTTTGTTCGGAT-3′; Integrated DNA Technologies). .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Isolation:

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. CHO-K1 cells were transiently transfected with either wt gAMN8-12 or del gAMN8-12 plasmids in duplicates and total RNA was isolated 48 hours after transfection (RNeasy Mini Kit; Qiagen, Ballerup, Denmark).

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA). .. Plasmids were isolated using the endotoxin-free ZR Plasmid Miniprep™-Classic kit (Zymo Research Corp., Irvine, CA) and quantified at 260 nm using a DS-11 spectrophotometer (DeNovix, Inc., Wilmington, DE).

    Article Title: A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture
    Article Snippet: Cross-linked plantlets were ground and nuclei were isolated and treated with SDS 0,3% at 65°C for 40min. .. Digestions were performed overnight at 37°C with 400U NheI-HF (NEB) for the IPT3 locus, 400U BglII (NEB) for the IPT7 locus or 400U MfeI (NEB) for the KRP7 locus.

    Flow Cytometry:

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Labeling:

    Article Title: Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila
    Article Snippet: 20 ug of DNA from each strain were digested with Nhe1 -HF (NEB R3131S, 25U) at 37C for overnight, and then separated using 4% DNA gel at 35V, 4°C for overnight. .. Dig-labeled Rab27 ORF probe was generated by PCR using the following primers: 5′-TTGACGTTGGCGCCGGTGCA-3′, 5′-TGAGCCTCTGCAATTAGCCGGAT-3′, labeled with Dig using Klenow (NEB) with labeling mix (NEB), boiled for 5 minutes to denature, and added to the membrane for hybridization overnight at 45°C.

    Cytometry:

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Detailed investigations of proximal tubular function in Imerslund-Gr?sbeck syndrome
    Article Snippet: Amplification products were cloned into expression vectors (pcDNA™ 3.1(+); Invitrogen, Taastrup, Denmark) using NheI-HF™ and Xho I restriction enzymes (New England Biolabs, Medinova Scientific A/S, Glostrup, Denmark) according to manufacturer’s guidelines (wt gAMN8-12 and del gAMN8-12) and subsequently transformed into competent cells (One Shot® TOP10; Invitrogen) for propagation. .. Splicing was analysed by RT-PCR (OneStep RT-PCR kit; Qiagen) using primers 5′-CACCTTCCTGGGTCTGCCTCAGTACC-3′ and 5′-GGCGCCACCAGCAGGACCAGCA-3′ according to manufacturer’s protocol. cDNA amplification products were cloned into vectors (pCR® II-TOPO® using TOPO TA cloning technique, Invitrogen) according to manufacturers protocol for subsequent sequencing.

    Gel Extraction:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: .. The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction. .. The final expression vectors for the fusion antibodies were constructed by in-frame ligation of the assembled DNA into the pFuse backbone (Invivogen) using T4 DNA ligase.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (catR0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.). .. The excised GFP sequence was replaced through restriction and ligation with T4 DNA ligase (cat#M0202L; New England Biolabs, Inc.) by the signal peptide of Tango1 (26 first amino acids, predicted by SignalP 4.1), which we PCR-amplified from pDONR221-Tango1 using primers that added the appropriate restriction sites (XbaISP-F: 5′-GGCTCTAGAATGCGGCTGACCAACGAGA-3′ and NheISP-R: 5′-CTAGCTAGCAGCCCACGTCAAAGTTGGAA-3′).

    Purification:

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Pre-mixed DNA linearized vector and PCR insert (1 μg μL−1 ) was electroporated into freshly prepare EBY100 competent cells, where the full constructs are reassembled via homologous recombination .

    Plasmid Preparation:

    Article Title: Rational design of a Kv1.3 channel-blocking antibody as a selective immunosuppressant
    Article Snippet: Paragraph title: Cloning of Antibody Expression Vector. ... The fusion gene fragments were assembled by overlap extension PCR and digested with the restriction enzymes EcoRI-HF and NheI-HF (New England Biolabs), followed by DNA gel extraction.

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were contransformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified before experiments.

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: .. In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA). .. The reporter constructs were then used to transform competent Escherichia coli DH5α cells (New England Biolabs) and plated on 100 μg/mL ampicillin Luria-Bertani (LB) agar plates.

    Article Title: Efficient derivation of knock-out and knock-in rats using embryos obtained by in vitro fertilization
    Article Snippet: Paragraph title: Targeting vector construction ... After confirmation of the sequence, homology arms and rTyr -repair cassette were excised using restriction endonucleases, Nhe I-HF and Mlu I-HF (New England Biolabs, Japan, Tokyo, Japan), and then ligated with pAAV_MCS2.

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.). .. The excised GFP sequence was replaced through restriction and ligation with T4 DNA ligase (cat#M0202L; New England Biolabs, Inc.) by the signal peptide of Tango1 (26 first amino acids, predicted by SignalP 4.1), which we PCR-amplified from pDONR221-Tango1 using primers that added the appropriate restriction sites (XbaISP-F: 5′-GGCTCTAGAATGCGGCTGACCAACGAGA-3′ and NheISP-R: 5′-CTAGCTAGCAGCCCACGTCAAAGTTGGAA-3′).

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were co-transformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified prior to experiments.

    Article Title: The Yin and Yang of SagS: Distinct Residues in the HmsP Domain of SagS Independently Regulate Biofilm Formation and Biofilm Drug Tolerance
    Article Snippet: .. Site-directed mutated variants of sagS were subsequently subcloned into the pMJT-1 vector using the oligonucleotides listed in in the supplemental material and the restriction enzymes NheI-HF and SacI-HF (New England Biolabs). .. Finally, the P. aeruginosa Δ sagS strain was complemented with wild-type sagS or each of the mutated versions of sagS in the pMJT1 vector.

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Pre-mixed DNA linearized vector and PCR insert (1 μg μL−1 ) was electroporated into freshly prepare EBY100 competent cells, where the full constructs are reassembled via homologous recombination .

    Positron Emission Tomography:

    Article Title: Engineered charge redistribution of Gp2 proteins through guided diversity for improved PET imaging of epidermal growth factor receptor
    Article Snippet: .. Gp2-encoding regions in DNA recovered from the final lysate-extracted EGFR flow cytometry sort were amplified by polymerase chain reaction, digested with NheI-HF and BamHI-HF restriction enzymes (New England Biolabs, Ipswich, MA), and ligated into a pET-22b vector containing a C-terminal hexa-histidine (Novagen, EMD Millipore, Billerica, MA) with T4 DNA ligase (New England Biolabs). .. Plasmids were transformed via heat-shock into T7 Express Competent E. coli (New England Biolabs) and proper transformants selected on lysogeny broth (LB) plates containing 50 mg/L kanamycin.

    Article Title: The Yin and Yang of SagS: Distinct Residues in the HmsP Domain of SagS Independently Regulate Biofilm Formation and Biofilm Drug Tolerance
    Article Snippet: The pET vector harboring the wild-type sagS gene was used as a template for construction of the mutants. .. Site-directed mutated variants of sagS were subsequently subcloned into the pMJT-1 vector using the oligonucleotides listed in in the supplemental material and the restriction enzymes NheI-HF and SacI-HF (New England Biolabs).

    Spectrophotometry:

    Article Title: Promoter Haplotypes of the ABCB1 Gene Encoding the P-Glycoprotein Differentially Affect Its Promoter Activity by Altering Transcription Factor Binding
    Article Snippet: In brief, constructs representing the four ABCB1 promoter haplotypes were generated by inserting the appropriate promoter sequences into the NanoLuc pNL1.1 vector (Promega) after double-digestion with the restriction enzymes Kpn I-HF and Nhe I-HF (New England Biolabs, Ipswich, MA). .. Plasmids were isolated using the endotoxin-free ZR Plasmid Miniprep™-Classic kit (Zymo Research Corp., Irvine, CA) and quantified at 260 nm using a DS-11 spectrophotometer (DeNovix, Inc., Wilmington, DE).

    Produced:

    Article Title: Tango1 spatially organizes ER exit sites to control ER export
    Article Snippet: This produced pTG-NheI-G-XhoI-SpeI-W, which contains two copies of GFP. .. The original GFP sequence in this plasmid was then excised by double digestion with XbaI (cat#R0145S; New England Biolabs, Inc.), for which a restriction site was already present between the UAS promoter and GFP in the original pTGW, and NheI (cat#R3131L; New England Biolabs, Inc.).

    FACS:

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: Two series of random mutagenesis and FACS-based selections (named I and II) were applied to improve both the binding affinity and crossreactivity of three promiscuous clones: CK1, CK2, and CK4. .. The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector.

    Homologous Recombination:

    Article Title: Nonspecificity in a nonimmune human scFv repertoire
    Article Snippet: .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were contransformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified before experiments.

    Article Title: Reduction of nonspecificity motifs in synthetic antibody libraries
    Article Snippet: .. The pCTCON2 vector was prepared for homologous recombination by digestion with SalI-HF followed by digestion with NheI-HF and BamI-HF (New England Biolabs). .. The two scFv fragments along with the digested vector were co-transformed into chemically competent yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research) and resultant clones were sequence verified prior to experiments.

    Article Title: Directed evolution of broadly crossreactive chemokine-blocking antibodies efficacious in arthritis
    Article Snippet: The mutagenized PCR products were further purified, re-amplified for additional 30 cycles in the absence of analog nucleotides, and combined with Sal I-HF, Nhe I-HF, and Bam HI-HI (New England BioLabs) digested pCT-CON vector. .. Pre-mixed DNA linearized vector and PCR insert (1 μg μL−1 ) was electroporated into freshly prepare EBY100 competent cells, where the full constructs are reassembled via homologous recombination .

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    New England Biolabs nhei hf
    Nhei Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhei hf/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    nhei hf - by Bioz Stars, 2020-04
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