esp 3i  (New England Biolabs)


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    Name:
    Esp3I
    Description:
    Esp3I 1 500 units
    Catalog Number:
    r0734l
    Price:
    343
    Size:
    1 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs esp 3i
    Esp3I
    Esp3I 1 500 units
    https://www.bioz.com/result/esp 3i/product/New England Biolabs
    Average 98 stars, based on 190 article reviews
    Price from $9.99 to $1999.99
    esp 3i - by Bioz Stars, 2020-09
    98/100 stars

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    1) Product Images from "Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59"

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.21.11065-11078.2002

    Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.
    Figure Legend Snippet: Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.

    Techniques Used: Marker, Recombinant, Sequencing, End-sequence Profiling

    Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.

    Techniques Used: Clone Assay, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Purification, End-sequence Profiling, Amplification

    Related Articles

    Transduction:

    Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
    Article Snippet: .. The double-stranded oligonucleotides were prepared and cloned into pL-CRISPR.SFFV.tRFP using Esp3I (isoschizomer of BsmBI, NEB) by conducting the cloning protocol previously described ( https://media.addgene.org/data/plasmids/57/57826/57826-attachment_n4-jQ6ZzmxPM.pdf), which produced the co-expression vector of sgRNA and SpCas9-P2A-TagRFP for transient transfection ( and ) or lentiviral transduction ( ). .. The NL4-3ΔEnv plasmid that contains a replication-defective molecular clone was acquired from the NIH AIDS reagent bank (catalog number: 11100).

    Clone Assay:

    Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
    Article Snippet: .. The double-stranded oligonucleotides were prepared and cloned into pL-CRISPR.SFFV.tRFP using Esp3I (isoschizomer of BsmBI, NEB) by conducting the cloning protocol previously described ( https://media.addgene.org/data/plasmids/57/57826/57826-attachment_n4-jQ6ZzmxPM.pdf), which produced the co-expression vector of sgRNA and SpCas9-P2A-TagRFP for transient transfection ( and ) or lentiviral transduction ( ). .. The NL4-3ΔEnv plasmid that contains a replication-defective molecular clone was acquired from the NIH AIDS reagent bank (catalog number: 11100).

    Transfection:

    Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
    Article Snippet: .. The double-stranded oligonucleotides were prepared and cloned into pL-CRISPR.SFFV.tRFP using Esp3I (isoschizomer of BsmBI, NEB) by conducting the cloning protocol previously described ( https://media.addgene.org/data/plasmids/57/57826/57826-attachment_n4-jQ6ZzmxPM.pdf), which produced the co-expression vector of sgRNA and SpCas9-P2A-TagRFP for transient transfection ( and ) or lentiviral transduction ( ). .. The NL4-3ΔEnv plasmid that contains a replication-defective molecular clone was acquired from the NIH AIDS reagent bank (catalog number: 11100).

    Amplification:

    Article Title: A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.
    Article Snippet: .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes.

    Ligation:

    Article Title: A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity
    Article Snippet: .. Golden Gate assembly [ ] was used in order to clone the double-stranded DNA Oligomers into the MLM3636 plasmid, using the following reaction: 150 ng MLM3636 plasmid 1 μL double stranded oligomer ligation mix 2 μL T4 ligase buffer (New England Biolabs) 13.25 μL ddH2 O 1 μL Esp3I (New England Biolabs) 1 μL T4 ligase (New England Biolabs) This reaction was put on a thermocycler using the following conditions: 37°C, 5 minutes 16°C, 10 minutes (10 cycles with these two steps) 37°C, 15 minutes 80°C, 5 minutes The ligated plasmid MLM3636(sgRNAmPrnp ) was subsequently transformed into DH5α chemically competent E . coli cells (Invitrogen) and plasmid purification was undertaken using Plasmid Maxi Kit (Qiagen). .. CAD5 cells were co-transfected using the MLM3636(sgRNAmPrnp ) plasmid and the hCas9 plasmid (hCas9 was a gift from George Church, Addgene plasmid # 41815, [ ]) dissolved in Lipofectamine 2000 (Invitrogen).

    Isolation:

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59
    Article Snippet: .. Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ). .. The MHV-A59 A clone was digested with Mlu I, which is located in the Topo II plasmid, treated with calf intestine alkaline phosphatase, and subsequently digested with Esp 3I.

    Purification:

    Article Title: A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.
    Article Snippet: .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes.

    Article Title: A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity
    Article Snippet: .. Golden Gate assembly [ ] was used in order to clone the double-stranded DNA Oligomers into the MLM3636 plasmid, using the following reaction: 150 ng MLM3636 plasmid 1 μL double stranded oligomer ligation mix 2 μL T4 ligase buffer (New England Biolabs) 13.25 μL ddH2 O 1 μL Esp3I (New England Biolabs) 1 μL T4 ligase (New England Biolabs) This reaction was put on a thermocycler using the following conditions: 37°C, 5 minutes 16°C, 10 minutes (10 cycles with these two steps) 37°C, 15 minutes 80°C, 5 minutes The ligated plasmid MLM3636(sgRNAmPrnp ) was subsequently transformed into DH5α chemically competent E . coli cells (Invitrogen) and plasmid purification was undertaken using Plasmid Maxi Kit (Qiagen). .. CAD5 cells were co-transfected using the MLM3636(sgRNAmPrnp ) plasmid and the hCas9 plasmid (hCas9 was a gift from George Church, Addgene plasmid # 41815, [ ]) dissolved in Lipofectamine 2000 (Invitrogen).

    Produced:

    Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
    Article Snippet: .. The double-stranded oligonucleotides were prepared and cloned into pL-CRISPR.SFFV.tRFP using Esp3I (isoschizomer of BsmBI, NEB) by conducting the cloning protocol previously described ( https://media.addgene.org/data/plasmids/57/57826/57826-attachment_n4-jQ6ZzmxPM.pdf), which produced the co-expression vector of sgRNA and SpCas9-P2A-TagRFP for transient transfection ( and ) or lentiviral transduction ( ). .. The NL4-3ΔEnv plasmid that contains a replication-defective molecular clone was acquired from the NIH AIDS reagent bank (catalog number: 11100).

    Concentration Assay:

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59
    Article Snippet: .. Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ). .. The MHV-A59 A clone was digested with Mlu I, which is located in the Topo II plasmid, treated with calf intestine alkaline phosphatase, and subsequently digested with Esp 3I.

    Polymerase Chain Reaction:

    Article Title: A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.
    Article Snippet: .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes.

    Sequencing:

    Article Title: A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.
    Article Snippet: .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes.

    Transformation Assay:

    Article Title: A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity
    Article Snippet: .. Golden Gate assembly [ ] was used in order to clone the double-stranded DNA Oligomers into the MLM3636 plasmid, using the following reaction: 150 ng MLM3636 plasmid 1 μL double stranded oligomer ligation mix 2 μL T4 ligase buffer (New England Biolabs) 13.25 μL ddH2 O 1 μL Esp3I (New England Biolabs) 1 μL T4 ligase (New England Biolabs) This reaction was put on a thermocycler using the following conditions: 37°C, 5 minutes 16°C, 10 minutes (10 cycles with these two steps) 37°C, 15 minutes 80°C, 5 minutes The ligated plasmid MLM3636(sgRNAmPrnp ) was subsequently transformed into DH5α chemically competent E . coli cells (Invitrogen) and plasmid purification was undertaken using Plasmid Maxi Kit (Qiagen). .. CAD5 cells were co-transfected using the MLM3636(sgRNAmPrnp ) plasmid and the hCas9 plasmid (hCas9 was a gift from George Church, Addgene plasmid # 41815, [ ]) dissolved in Lipofectamine 2000 (Invitrogen).

    End-sequence Profiling:

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59
    Article Snippet: .. Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ). .. The MHV-A59 A clone was digested with Mlu I, which is located in the Topo II plasmid, treated with calf intestine alkaline phosphatase, and subsequently digested with Esp 3I.

    Plasmid Preparation:

    Article Title: Fine-mapping within eQTL credible intervals by expression CROP-seq
    Article Snippet: .. The CROPseq-Guide-Puro plasmid [ ] (Addgene, Watertown MA, catalog number #86708, originally from Christoph Bock’s lab) was digested by Esp3I (NEB, R0734S). .. For each designed gRNA sequence, a pair of annealed oligos was cloned into the vector before the gRNA scaffold and after the U6 promoter.

    Article Title: Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites
    Article Snippet: .. The double-stranded oligonucleotides were prepared and cloned into pL-CRISPR.SFFV.tRFP using Esp3I (isoschizomer of BsmBI, NEB) by conducting the cloning protocol previously described ( https://media.addgene.org/data/plasmids/57/57826/57826-attachment_n4-jQ6ZzmxPM.pdf), which produced the co-expression vector of sgRNA and SpCas9-P2A-TagRFP for transient transfection ( and ) or lentiviral transduction ( ). .. The NL4-3ΔEnv plasmid that contains a replication-defective molecular clone was acquired from the NIH AIDS reagent bank (catalog number: 11100).

    Article Title: A sort-seq approach to the development of single fluorescent protein biosensors
    Article Snippet: .. In a 10μL reaction containing 13pmol of the PyronicSF linker library in the holding plasmid, 13pmol of EMMA-attB-Dest, 5 units Esp3I (NEB), 100 units T4 DNA ligase (NEB), and 1X T4 DNA ligase buffer (NEB). .. The Golden Gate reaction was cleaned using a DNA Clean & Concentrator-5 Kit, eluted in 6μL of water and transformed by electroporation using 1μL reaction added to 25μL of E. cloni 10G ELITE cells.

    Article Title: A One-Step Process for the Construction of Phage Display scFv and VHH Libraries.
    Article Snippet: .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes. .. In this work we present a one-step cloning approach for the establishment of antibody phage display libraries relying on type IIs restriction enzymes.

    Article Title: Multimodal Analysis of Composition and Spatial Architecture in Human Squamous Cell Carcinoma
    Article Snippet: .. 16ng of the oligo pool was then assembled with 446ng Esp3I digested pLentiguide vector in a Gibson Assembly reaction (NEB). .. The reaction was transformed into Stellar competent cells and grown overnight in liquid culture with ampicillin selection.

    Article Title: A bispecific immunotweezer prevents soluble PrP oligomers and abolishes prion toxicity
    Article Snippet: .. Golden Gate assembly [ ] was used in order to clone the double-stranded DNA Oligomers into the MLM3636 plasmid, using the following reaction: 150 ng MLM3636 plasmid 1 μL double stranded oligomer ligation mix 2 μL T4 ligase buffer (New England Biolabs) 13.25 μL ddH2 O 1 μL Esp3I (New England Biolabs) 1 μL T4 ligase (New England Biolabs) This reaction was put on a thermocycler using the following conditions: 37°C, 5 minutes 16°C, 10 minutes (10 cycles with these two steps) 37°C, 15 minutes 80°C, 5 minutes The ligated plasmid MLM3636(sgRNAmPrnp ) was subsequently transformed into DH5α chemically competent E . coli cells (Invitrogen) and plasmid purification was undertaken using Plasmid Maxi Kit (Qiagen). .. CAD5 cells were co-transfected using the MLM3636(sgRNAmPrnp ) plasmid and the hCas9 plasmid (hCas9 was a gift from George Church, Addgene plasmid # 41815, [ ]) dissolved in Lipofectamine 2000 (Invitrogen).

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    New England Biolabs esp 3i
    Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 <t>Esp</t> 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.
    Esp 3i, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/esp 3i/product/New England Biolabs
    Average 98 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    esp 3i - by Bioz Stars, 2020-09
    98/100 stars
      Buy from Supplier

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    Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.

    Journal: Journal of Virology

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

    doi: 10.1128/JVI.76.21.11065-11078.2002

    Figure Lengend Snippet: Identification of marker mutations in recombinant virus. Various icMHV-A59#1 or wild-type virus amplicons were subcloned into Topo II vectors and sequenced. (A) icMHV-A59#1 sequence across the mutated 3512 Esp 3I site; (B) icMHV-A59#1 sequence across the mutated 48 Esp 3I site and the MHV A/B junction; (C) icMHV-A59#1 sequence across the mutated 17 Esp 3I site; (D) icMHV-A59#1 sequence across the Rsr II site and MHV F/G junction; (E) wild-type MHV-A59 sequence across the same domain as shown in panel D. Overlined sequences include the mutated restriction sites and the corresponding wild-type nucleotides at these positions. In panels B and D, the bracketed sequences represent the MHV A/B and F/G junction domains, respectively. Note that all evidence of the Esp 3I site, which had been engineered into the sequence, is gone.

    Article Snippet: Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ).

    Techniques: Marker, Recombinant, Sequencing, End-sequence Profiling

    Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.

    Journal: Journal of Virology

    Article Title: Systematic Assembly of a Full-Length Infectious cDNA of Mouse Hepatitis Virus Strain A59

    doi: 10.1128/JVI.76.21.11065-11078.2002

    Figure Lengend Snippet: Restriction fragment length polymorphism analysis of molecularly cloned virus. Cultures were infected with MHV-A59 or icMHV-A59#1, and intracellular RNA was isolated at 8 h p.i. With primer pairs and RT-PCR, cDNA amplicons were isolated that contained the various marker mutations that had been inserted into the component clones. The purified wild-type MHV-A59 and icMHV-A59#1 amplicons were restricted with Esp 3I or Rsr II and separated in 0.8% agarose gels. Lane 1, wild-type A59 amplicon from nucleotides 2020 to 5031, uncut; lane 2, wild-type A59 amplicon from nucleotides 2020 to 5031 digested with Esp 3I; lane 3, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, uncut; lane 4, icMHV-A59#1 amplicon from nucleotides 2020 to 5031, Esp 3I digested; lane 5, wild-type A59 amplicon from nucleotides 16351 to 17875, uncut; lane 6, wild-type A59 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 7, icMHV-A59#1 amplicon from nucleotides 16351 to 17875, uncut; lane 8, icMHV-A59#1 amplicon from nucleotides 16351 to 17875 restricted with Esp 3I; lane 9, wild-type A59 amplicon from nucleotides 22060 to 25416, uncut; lane 10, wild-type A59 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 11, icMHV-A59#1 amplicon from nucleotides 22060 to 25416, uncut; lane 12, icMHV-A59#1 amplicon from nucleotides 22060 to 25416 restricted with Rsr II; lane 13, 1-kb ladder.

    Article Snippet: Each of the plasmids was grown to a high concentration, isolated, and digested, or double digested, with Esp 3I, Bgl I, or Not I according to the manufacturer's directions (New England Biolabs) (Fig. ).

    Techniques: Clone Assay, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Marker, Purification, End-sequence Profiling, Amplification