r0131  (New England Biolabs)


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    Name:
    NheI
    Description:
    NheI 5 000 units
    Catalog Number:
    R0131L
    Price:
    282
    Category:
    Restriction Enzymes
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    5 000 units
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    Structured Review

    New England Biolabs r0131
    NheI
    NheI 5 000 units
    https://www.bioz.com/result/r0131/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r0131 - by Bioz Stars, 2021-05
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    Related Articles

    Plasmid Preparation:

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae
    Article Snippet: The uracil independent and ampicillin resistance pJRoC30 vector was obtained from the California Institute of Technology (CALTECH). .. The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs. ..

    Article Title: Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene
    Article Snippet: Construction of Minigenes A plasmid, pcDNA3.1/Hygro(+) encoding drug resistant genes as well as CMV promoter and BGH-poly A signal was obtained from Invitrogen (V87020, thermo fisher scientific, Waltham, MA, USA). .. In this plasmid, the synthesized DNA was inserted after digestion with restriction enzymes, Nhe I and Xho I (R0131S and R0146S, New England Biolabs, Ipswich, MA, USA). .. The insert was designed with the following sequences from the 5′ to 3′ ends in this order; Nhe I restriction enzyme recognition site, mCherry, 5′ part of eGFP, an artificial intron consisting of the 5′-end of DMD intron 18, the multicloning site sequence (MCS), the 3′-end of DMD intron 19, 3′ part of eGFP, and Xho I restriction enzyme recognition site ( A).

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: Two single-stranded oligonucleotides surrounding the AvrII sites in pKFAV4 were synthesized ( ) and fused to form a double stranded DNA (AvrII-PacI, 96 bp in length) through DNA polymerasemediated extension reaction (Q5 High-Fidelity DNA Polymerase, Cat. M0491S; New England Biolabs, Ipswich, Massachusetts, USA), and a PacI site (ttaattaa) was added in the middle to demarcate the two AvrII-centered parts. pKFAV4 was digested with AvrII, the small fragment (16.7 kb) was recovered from agarose gel (Zymoclean Large Fragment DNA Recovery Kit, Cat.D4045; Zymo Research, Irvine, CA, USA) and mixed with AvrII-PacI fragment, and Gibson assembly was performed to generate an intermediate plasmid pKFAV4AP ( pKFAV4 A vrII- P acI) (NEBuilderHiFi DNA Assembly Master Mix, Cat. E2621; New England Biolabs). .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: 2.8 Preparation of a Human Full-length CD22 and CD22ΔE12–14 Plasmids The cDNA fragments encoding either full-length CD22 (CD22FL) or truncated CD22 lacking exons 12–14 (CD22ΔE12–14) were generated by PCR amplification using the Phusion High Fidelity PCR Kit (New England Biolabs, catalog no. E0553L) with the following primer sets: full-length Fwd/Rev 5′-CTTGGT GCTAGC ATGCATCTCCTCGGC-3′/5′CCGG TCTCGAG GATGTTTGAGGATCACATAGTC-3′, and truncation ΔE12–14 Fwd/Rev 5′-CTTGGT GCTAGC ATGCATCTCCTCGGC-3′/5′-CCGGT CTCGAG CCTTTTTATTCCTCAC-3′. .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells
    Article Snippet: The recombinant plasmids were detected by restriction digestion with NheI and XhoI enzymes (New England BioLabs, USA) and confirmed by sequence analysis (Bioneer, Korea). .. Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours. .. After the enzymes thermal inactivation, the products of digestion were analyzed by electrophoresis on 1% agarose low melting point gel (Roche, Germany) and bands of fliC fragments and digested pVAX1 plasmids were purified from the agarose gel by the Agarose Gel DNA Extraction Kit (Roche, Germany).

    Recombinant:

    Article Title: Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells
    Article Snippet: After selecting recombinant clones, the plasmid DNA was extracted from cells cultured overnight with shaking at 250 rpm at 37°C by using the Miniprep plasmid purification kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. .. The selected recombinant plasmids were verified by restriction digestion with NheI and XhoI restriction enzymes (New England BioLabs, Ipswich, MA, USA) and sequenced using M13 primers. .. Subcloning of Surface Antigen 1 Gene The pPrime vector carrying the SAG1 gene and the eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, USA) were digested by NdeI and XhoI restriction enzymes.

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells
    Article Snippet: The recombinant plasmids were detected by restriction digestion with NheI and XhoI enzymes (New England BioLabs, USA) and confirmed by sequence analysis (Bioneer, Korea). .. Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours. .. After the enzymes thermal inactivation, the products of digestion were analyzed by electrophoresis on 1% agarose low melting point gel (Roche, Germany) and bands of fliC fragments and digested pVAX1 plasmids were purified from the agarose gel by the Agarose Gel DNA Extraction Kit (Roche, Germany).

    Synthesized:

    Article Title: Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene
    Article Snippet: Construction of Minigenes A plasmid, pcDNA3.1/Hygro(+) encoding drug resistant genes as well as CMV promoter and BGH-poly A signal was obtained from Invitrogen (V87020, thermo fisher scientific, Waltham, MA, USA). .. In this plasmid, the synthesized DNA was inserted after digestion with restriction enzymes, Nhe I and Xho I (R0131S and R0146S, New England Biolabs, Ipswich, MA, USA). .. The insert was designed with the following sequences from the 5′ to 3′ ends in this order; Nhe I restriction enzyme recognition site, mCherry, 5′ part of eGFP, an artificial intron consisting of the 5′-end of DMD intron 18, the multicloning site sequence (MCS), the 3′-end of DMD intron 19, 3′ part of eGFP, and Xho I restriction enzyme recognition site ( A).

    Polymerase Chain Reaction:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: Two single-stranded oligonucleotides surrounding the AvrII sites in pKFAV4 were synthesized ( ) and fused to form a double stranded DNA (AvrII-PacI, 96 bp in length) through DNA polymerasemediated extension reaction (Q5 High-Fidelity DNA Polymerase, Cat. M0491S; New England Biolabs, Ipswich, Massachusetts, USA), and a PacI site (ttaattaa) was added in the middle to demarcate the two AvrII-centered parts. pKFAV4 was digested with AvrII, the small fragment (16.7 kb) was recovered from agarose gel (Zymoclean Large Fragment DNA Recovery Kit, Cat.D4045; Zymo Research, Irvine, CA, USA) and mixed with AvrII-PacI fragment, and Gibson assembly was performed to generate an intermediate plasmid pKFAV4AP ( pKFAV4 A vrII- P acI) (NEBuilderHiFi DNA Assembly Master Mix, Cat. E2621; New England Biolabs). .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: A rationally designed nanoparticle for RNA interference therapy in B-lineage lymphoid malignancies
    Article Snippet: 2.8 Preparation of a Human Full-length CD22 and CD22ΔE12–14 Plasmids The cDNA fragments encoding either full-length CD22 (CD22FL) or truncated CD22 lacking exons 12–14 (CD22ΔE12–14) were generated by PCR amplification using the Phusion High Fidelity PCR Kit (New England Biolabs, catalog no. E0553L) with the following primer sets: full-length Fwd/Rev 5′-CTTGGT GCTAGC ATGCATCTCCTCGGC-3′/5′CCGG TCTCGAG GATGTTTGAGGATCACATAGTC-3′, and truncation ΔE12–14 Fwd/Rev 5′-CTTGGT GCTAGC ATGCATCTCCTCGGC-3′/5′-CCGGT CTCGAG CCTTTTTATTCCTCAC-3′. .. The correct PCR products (FL: 2541-bp and ΔE12–14: 2208-bp) were ligated into the 8497-bp lentiviral vector pCL6-2AEGwo through the NheI and XhoI restriction sites (underlined) using the Quick Ligase kit (New England Biolabs catalog no. M2200L) following the manufacturer's instructions. .. The pCL6-2AEGwo lentiviral backbone vector, a kind gift from Dr. Zanxian Xia, School of Biological Science and Technology, Central South University, Changsha, Hunan 410078, China, contains both a “ribosome-skip” fragment encoding the 2A-like peptide APVKQTLNFDLLKLAGDVESNPGP and an in-frame eGFP fluorescent coding sequence downstream of a multiple cloning site.

    Expressing:

    Article Title: User-Friendly Reverse Genetics System for Modification of the Right End of Fowl Adenovirus 4 Genome
    Article Snippet: Two single-stranded oligonucleotides surrounding the AvrII sites in pKFAV4 were synthesized ( ) and fused to form a double stranded DNA (AvrII-PacI, 96 bp in length) through DNA polymerasemediated extension reaction (Q5 High-Fidelity DNA Polymerase, Cat. M0491S; New England Biolabs, Ipswich, Massachusetts, USA), and a PacI site (ttaattaa) was added in the middle to demarcate the two AvrII-centered parts. pKFAV4 was digested with AvrII, the small fragment (16.7 kb) was recovered from agarose gel (Zymoclean Large Fragment DNA Recovery Kit, Cat.D4045; Zymo Research, Irvine, CA, USA) and mixed with AvrII-PacI fragment, and Gibson assembly was performed to generate an intermediate plasmid pKFAV4AP ( pKFAV4 A vrII- P acI) (NEBuilderHiFi DNA Assembly Master Mix, Cat. E2621; New England Biolabs). .. Delete ORF1-ORF2 in pKFAV4 pKFAV4AP was digested with AgeI/NheI, and the fragment of 4942 bp was recovered; PCR was performed to amplify AgeI-ORF1 fragment (Open Reading Frame, ORF; from upstream of AgeI site in ORF0 to ORF1; 204 bp) with primers of 1707KFAV4AgeIF/R ( ) using pKFAV4AP as the template (Q5 High-Fidelity DNA Polymerase); PCR was performed to amplify GFP expression cassette (1672 bp) with primers of 1707F02GFPF/R using pAd5GFP as the template; and these three fragments were fused together to generate plasmid pKFAV4APN-GFP ( pKFAV4AP N heI -GFP ) by DNA assembly. pKFAV4APN-GFP was digested with NheI, treated with CIP (Calf Intestinal Alkaline Phosphatase; New England Biolabs), and ligated with the large fragment of NheI-digested pKFAV4AP (9840 bp) to generate plasmid pKFAV4AP-GFP (16,575 bp). pKFAV4AP-GFP was digested with PacI and fused to the large fragment of AvrII-digested pKFAV4 (29,468 bp) to generate pKFAV4-GFP (45,981 bp) by DNA assembly (NEBuilderHiFi DNA Assembly Master Mix). .. Replace Coding Sequence of FAdV-4 ORF4 with That of mCherry in pKFAV4-GFP PvuI and BamHI were single cutters flanking ORF4 coding sequence (CDS) in pKFAV4APN-GFP.

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells
    Article Snippet: The recombinant plasmids were detected by restriction digestion with NheI and XhoI enzymes (New England BioLabs, USA) and confirmed by sequence analysis (Bioneer, Korea). .. Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours. .. After the enzymes thermal inactivation, the products of digestion were analyzed by electrophoresis on 1% agarose low melting point gel (Roche, Germany) and bands of fliC fragments and digested pVAX1 plasmids were purified from the agarose gel by the Agarose Gel DNA Extraction Kit (Roche, Germany).

    Subcloning:

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells
    Article Snippet: The recombinant plasmids were detected by restriction digestion with NheI and XhoI enzymes (New England BioLabs, USA) and confirmed by sequence analysis (Bioneer, Korea). .. Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours. .. After the enzymes thermal inactivation, the products of digestion were analyzed by electrophoresis on 1% agarose low melting point gel (Roche, Germany) and bands of fliC fragments and digested pVAX1 plasmids were purified from the agarose gel by the Agarose Gel DNA Extraction Kit (Roche, Germany).

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    New England Biolabs nhei
    Restriction Enzyme Analysis of Recombinant pVAX1-SAG1 Line 1 and 2, the recombinant pVAX-SAG1 plasmid digested by <t>NheI</t> and <t>XhoI;</t> Line M, 1 kb DNA ladder.
    Nhei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhei/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nhei - by Bioz Stars, 2021-05
    99/100 stars
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    Restriction Enzyme Analysis of Recombinant pVAX1-SAG1 Line 1 and 2, the recombinant pVAX-SAG1 plasmid digested by NheI and XhoI; Line M, 1 kb DNA ladder.

    Journal: Jundishapur Journal of Microbiology

    Article Title: Cloning and Expression of Major Surface Antigen 1 Gene of Toxoplasma gondii RH Strain Using the Expression Vector pVAX1 in Chinese Hamster Ovary Cells

    doi: 10.5812/jjm.22570

    Figure Lengend Snippet: Restriction Enzyme Analysis of Recombinant pVAX1-SAG1 Line 1 and 2, the recombinant pVAX-SAG1 plasmid digested by NheI and XhoI; Line M, 1 kb DNA ladder.

    Article Snippet: The selected recombinant plasmids were verified by restriction digestion with NheI and XhoI restriction enzymes (New England BioLabs, Ipswich, MA, USA) and sequenced using M13 primers.

    Techniques: Recombinant, Plasmid Preparation

    Dual fluorescence-based splicing reporter minigenes. ( A ) The structure of a ready-made dual fluorescence-based splicing reporter minigene is schematically described. An expression vector pcDNA3.1/Hygro(+) encoding the CMV promoter (CMV) and the polyadenylation signal of bovine growth hormone gene (poly A) was inserted with the synthesized 1683 bp long fragment with the use of Nhe I and Xho I restriction enzymes. The synthesized DNA comprised of sequences of the Nhe I restriction enzyme recognition site, mCherry, the 5′ region of eGFP (5′-eGFP), the 5′-end of DMD intron 18, the multicloning site (MCS), the 3′-end of DMD intron 19, the 3′ region of eGFP (3′-eGFP), and the Xho I restriction enzyme recognition site. The MCS sequence is immediately flanked upstream and downstream by the first part of DMD intron18 and the last part of DMD intron19, respectively. The original construct named as FM was modified to create FMv2 by replacing 15 nucleotides. ( B ) The ready-made minigene (FMv2) was modified to produce FMv2 CD44 v8 as follows. PCR amplified genomic region of exon v8 of the CD44 gene was inserted into the MCS of FMv2 using Hind III and BamH I restriction enzymes. The sequence of the MCS is described in the middle together with restriction enzyme recognition sites. Exon v8 of the CD44 gene (102 bp) and its flanking introns (505 and 504 bps, respectively) were amplified using primers (horizontal arrowhead) with Hind III and BamH I sequences added at the 5′end.

    Journal: International Journal of Molecular Sciences

    Article Title: Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

    doi: 10.3390/ijms21239136

    Figure Lengend Snippet: Dual fluorescence-based splicing reporter minigenes. ( A ) The structure of a ready-made dual fluorescence-based splicing reporter minigene is schematically described. An expression vector pcDNA3.1/Hygro(+) encoding the CMV promoter (CMV) and the polyadenylation signal of bovine growth hormone gene (poly A) was inserted with the synthesized 1683 bp long fragment with the use of Nhe I and Xho I restriction enzymes. The synthesized DNA comprised of sequences of the Nhe I restriction enzyme recognition site, mCherry, the 5′ region of eGFP (5′-eGFP), the 5′-end of DMD intron 18, the multicloning site (MCS), the 3′-end of DMD intron 19, the 3′ region of eGFP (3′-eGFP), and the Xho I restriction enzyme recognition site. The MCS sequence is immediately flanked upstream and downstream by the first part of DMD intron18 and the last part of DMD intron19, respectively. The original construct named as FM was modified to create FMv2 by replacing 15 nucleotides. ( B ) The ready-made minigene (FMv2) was modified to produce FMv2 CD44 v8 as follows. PCR amplified genomic region of exon v8 of the CD44 gene was inserted into the MCS of FMv2 using Hind III and BamH I restriction enzymes. The sequence of the MCS is described in the middle together with restriction enzyme recognition sites. Exon v8 of the CD44 gene (102 bp) and its flanking introns (505 and 504 bps, respectively) were amplified using primers (horizontal arrowhead) with Hind III and BamH I sequences added at the 5′end.

    Article Snippet: In this plasmid, the synthesized DNA was inserted after digestion with restriction enzymes, Nhe I and Xho I (R0131S and R0146S, New England Biolabs, Ipswich, MA, USA).

    Techniques: Fluorescence, Expressing, Plasmid Preparation, Synthesized, Sequencing, Construct, Modification, Polymerase Chain Reaction, Amplification

    Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Journal: Bioengineered

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    doi: 10.4161/bioe.29167

    Figure Lengend Snippet: Figure 4. In vivo assembly of synthetic genes by IVOE. Each overlapping region allowed crossover events to occur between fragments giving rise to an autonomously repaired vector containing single and double expression cassettes in the correct orientation. ( A ) and ( B ) Single expression cassettes are constructed in pESC by engineering specific primers containing overhangs to foster in vivo cloning with the linearized plasmid in yeast (pJRoC30 was used as template for Vp or Lac amplifications). ( C ) and ( D ) The pESC constructs obtained in ( A ) and ( B ) were used as scaffolds to assemble Lac and Vp genes under the control of different promoter/terminator pairs. Primers used: (1)-MCS1- Vp / Lac -α-BamHI, (2)-MCS2- Vp -ter-NheI, (3)-MCS2- Lac -ter-NheI, (4)-MCS1- Vp / Lac -α-SpeI, (5)-MCS1- Lac -ter-SacI and (6)-MCS1- Vp -ter-SacI. Black arrows indicate the direction of the transcription process.

    Article Snippet: The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs.

    Techniques: In Vivo, Plasmid Preparation, Expressing, Construct, Clone Assay

    Electrophoresis of pPrime- fliC Recombinant Plasmids From White Colonies on 1% Agarose Gel Lane 1 to 6: extracted Pprime- fliC plasmids; Lane 8 to 13: Pprime- fliC plasmids digested by XhoI; Lane 15 to 20: Pprime- fliC plasmids digested by XhoI and NheI; Lane 7 and 14: 1 Kbp DNA ladder vivantis.

    Journal: Jundishapur Journal of Microbiology

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells

    doi: 10.5812/jjm.12351

    Figure Lengend Snippet: Electrophoresis of pPrime- fliC Recombinant Plasmids From White Colonies on 1% Agarose Gel Lane 1 to 6: extracted Pprime- fliC plasmids; Lane 8 to 13: Pprime- fliC plasmids digested by XhoI; Lane 15 to 20: Pprime- fliC plasmids digested by XhoI and NheI; Lane 7 and 14: 1 Kbp DNA ladder vivantis.

    Article Snippet: Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours.

    Techniques: Electrophoresis, Recombinant, Agarose Gel Electrophoresis

    AgaroseGel Electrophoresis of Restriction Enzyme Digestion of Purified pVAX- fliC Recombinant Plasmid Lane 1: pVAX- fliC plasmid digested by XhoI had one band (4419 bp); Lane 2: Double Digestion by NheI and XhoI on pVAX- fliC plasmid had two bands that were 1509 bp (down) and 2910 bp (up); Lane 3: 1 Kbp DNA ladder Vivantis.

    Journal: Jundishapur Journal of Microbiology

    Article Title: Cloning of fliC Gene From Salmonella typhimurium in the Expression Vector pVAX1 and Evaluation of its Expression in Eukaryotic Cells

    doi: 10.5812/jjm.12351

    Figure Lengend Snippet: AgaroseGel Electrophoresis of Restriction Enzyme Digestion of Purified pVAX- fliC Recombinant Plasmid Lane 1: pVAX- fliC plasmid digested by XhoI had one band (4419 bp); Lane 2: Double Digestion by NheI and XhoI on pVAX- fliC plasmid had two bands that were 1509 bp (down) and 2910 bp (up); Lane 3: 1 Kbp DNA ladder Vivantis.

    Article Snippet: Subcloning in pVAX1 Eukaryotic Expression Vector The recombinant plasmid pPrime-fliC and eukaryotic expression vector pVAX1 (Invitrogen, Carlsbad, CA, USA) were digested by XhoI and NheI enzymes (New England BioLabs, USA) at 37˚C for 16 hours.

    Techniques: Electrophoresis, Purification, Recombinant, Plasmid Preparation