sali treatment  (New England Biolabs)


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    Name:
    SalI
    Description:
    SalI 10 000 units
    Catalog Number:
    r0138l
    Price:
    261
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs sali treatment
    SalI
    SalI 10 000 units
    https://www.bioz.com/result/sali treatment/product/New England Biolabs
    Average 94 stars, based on 19442 article reviews
    Price from $9.99 to $1999.99
    sali treatment - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) Product Images from "Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion"

    Article Title: Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw861

    Bead separation from the tethered DNA substrate containing attB-attP by addition of SalI. ( A ) In the schematic representation of the 1303 bp recombination substrates (containing attB-attP or attL-attR ), the integrase cleavage sites are indicated by the short vertical arrows. The vertical line at the left and the sphere at the right indicate the tethering glass surface and the polystyrene bead, respectively. Double strand cleavage at one or both att sites will result in bead detachment from the tethered DNA. The number of molecules with detached beads following SDS treatment from three assays are tabulated. The orientation of the att sites in a substrate is indicated by the arrows. The attB-attP reactions contained integrase. The attL-attR reaction contained integrase plus gp3. ( B ) In the 1303 bp DNA substrate with head-to-tail attB-attP sites, the SalI recognition site located 241 bp away from the end attached to the glass surface. SalI digestion would release the bead along with the long DNA fragment from the short fragment that remains tethered. The BM amplitude time trace of a molecule before and after SalI addition (indicated by the short vertical arrow) are shown. The abrupt termination of the trace resulted from bead detachment. Such events from three separate experiments are tabulated. N refers to the number of DNA molecules observed.
    Figure Legend Snippet: Bead separation from the tethered DNA substrate containing attB-attP by addition of SalI. ( A ) In the schematic representation of the 1303 bp recombination substrates (containing attB-attP or attL-attR ), the integrase cleavage sites are indicated by the short vertical arrows. The vertical line at the left and the sphere at the right indicate the tethering glass surface and the polystyrene bead, respectively. Double strand cleavage at one or both att sites will result in bead detachment from the tethered DNA. The number of molecules with detached beads following SDS treatment from three assays are tabulated. The orientation of the att sites in a substrate is indicated by the arrows. The attB-attP reactions contained integrase. The attL-attR reaction contained integrase plus gp3. ( B ) In the 1303 bp DNA substrate with head-to-tail attB-attP sites, the SalI recognition site located 241 bp away from the end attached to the glass surface. SalI digestion would release the bead along with the long DNA fragment from the short fragment that remains tethered. The BM amplitude time trace of a molecule before and after SalI addition (indicated by the short vertical arrow) are shown. The abrupt termination of the trace resulted from bead detachment. Such events from three separate experiments are tabulated. N refers to the number of DNA molecules observed.

    Techniques Used:

    Related Articles

    DNA Extraction:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Clone Assay:

    Article Title: Self-reactive IgE exacerbates interferon responses associated with autoimmunity
    Article Snippet: .. To generate the human dsDNA-specific IgE (IgED ), E11 variable regions were cloned by restriction digestion using BssHII and SalI enzymes from New England Biolabs and ligated into an IgE Orip/EBNA-1-based episomal mammalian expression vector pOE (MedImmune LLC). .. A human IgE specific for metapneumovirus (hMPV) was generated in a similar fashion to be used as an isotype control (IgEI ).

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Amplification:

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Article Title: Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases
    Article Snippet: .. After amplification, the PCR product was digested with PciI and SalI restriction enzymes and ligated using T4 DNA ligase (NEB) into the vector pET28a (Novagen), which had been digested with the restriction enzymes NcoI and XhoI to create compatible sticky ends. .. The ligated vector was transformed into DH5α competent cells, and clones from the resulting colonies were verified by sequencing.

    Reporter Assay:

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Concentration Assay:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Mutagenesis:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Subcloning:

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Spectrophotometry:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Electroporation Bacterial Transformation:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Polymerase Chain Reaction:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Article Title: Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22
    Article Snippet: .. The resulting PCR product was restriction digested with EcoRI and SalI enzymes and ligated into pMAL-c2X vector (NEB) cut with the same enzymes. .. The MBP-L22 sequence was confirmed by DNA sequencing (Keck Facility).

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Article Title: Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases
    Article Snippet: .. After amplification, the PCR product was digested with PciI and SalI restriction enzymes and ligated using T4 DNA ligase (NEB) into the vector pET28a (Novagen), which had been digested with the restriction enzymes NcoI and XhoI to create compatible sticky ends. .. The ligated vector was transformed into DH5α competent cells, and clones from the resulting colonies were verified by sequencing.

    Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis-eQTL
    Article Snippet: .. The restriction enzymes SalI and KpnI (NEB) were used for restriction digestion on PCR products and for the pCMV-N-FLAG (Takara) vector having similar restriction sites. .. CA8-204C was produced using GENEART site-directed mutagenesis system (Invitrogen Life Technologies, Carlsbad, CA) and specific primers (forward: 5’-GTTGGATTCAGTCCACGTCTTGATGTTATTT-3’, reverse: 5’-AAATAACATCAAGACGTGGACTGAATCCAAC-3’) were employed to create one nucleotide substitution at position 1416 in the CA8-204 transcript.

    Generated:

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Luciferase:

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Introduce:

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Expressing:

    Article Title: Self-reactive IgE exacerbates interferon responses associated with autoimmunity
    Article Snippet: .. To generate the human dsDNA-specific IgE (IgED ), E11 variable regions were cloned by restriction digestion using BssHII and SalI enzymes from New England Biolabs and ligated into an IgE Orip/EBNA-1-based episomal mammalian expression vector pOE (MedImmune LLC). .. A human IgE specific for metapneumovirus (hMPV) was generated in a similar fashion to be used as an isotype control (IgEI ).

    Modification:

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Binding Assay:

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    other:

    Article Title: Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion
    Article Snippet: SalI treatment of tethered DNA molecules The DNA molecules were tethered to the glass surface in the reaction chamber containing the SalI digestion buffer (from New England BioLabs).

    Plasmid Preparation:

    Article Title: CRISPR-Cas9-guided Genome Engineering in C. elegans
    Article Snippet: .. Kpn I (NEB R0142S) Sal I (NEB R0138S) pUC19 (NEB N3041S) pPV477 (Addgene plasmid #42930) 37°C water bath incubator 42°C water bath incubator for bacterial transformation Spectrophotometer for measuring DNA concentration PCR thermo cycler (Biorad T100 or equivalent) LB agar plate containing 100 μ g/ml ampicillin (UNIT 1.1) LB liquid medium containing 100 μg/ml ampicillin (UNIT 1.1) Gibson assembly Master Mix (NEB E2611S) High Fidelity Phusion DNA polymerase (NEB M0530S or equivalent) Plasmid Miniprep Kit (GeneJet K0502 or Qiagen 27104) Plasmid Midiprep Kit (Qiagen 12143) Nuclease-free water (Qiagen 129114 or equivalent) Gel DNA Extraction Kit (Zymoclean D4001) Q5® Site-Directed Mutagenesis Kit (NEB E0554S) UP-F : 5’-ACGGCCAGTGAATTCGAGCTCGGTA + ~N18-24 -3’. .. N18-24 from upstream of a gene of interest UP-R : 5’-GTGAAAAGTTCTTCTCCTTTACTCAT + ~N18-24 (RC) -3’.

    Article Title: Multiple domains of EBER 1, an Epstein-Barr virus noncoding RNA, recruit human ribosomal protein L22
    Article Snippet: .. The resulting PCR product was restriction digested with EcoRI and SalI enzymes and ligated into pMAL-c2X vector (NEB) cut with the same enzymes. .. The MBP-L22 sequence was confirmed by DNA sequencing (Keck Facility).

    Article Title: Self-reactive IgE exacerbates interferon responses associated with autoimmunity
    Article Snippet: .. To generate the human dsDNA-specific IgE (IgED ), E11 variable regions were cloned by restriction digestion using BssHII and SalI enzymes from New England Biolabs and ligated into an IgE Orip/EBNA-1-based episomal mammalian expression vector pOE (MedImmune LLC). .. A human IgE specific for metapneumovirus (hMPV) was generated in a similar fashion to be used as an isotype control (IgEI ).

    Article Title: Enhanced Control of Oncolytic Measles Virus Using MicroRNA Target Sites
    Article Snippet: .. Finally, the modified N gene was excised with MluI and SbfI and inserted into the pMV-EGFP vector digested with MluI and SbfI to generate pMV-EGFP-NmiRTS7 . pMV-EGFP-FmiRTS7 and -HmiRTS7 were generated by insertion of custom-designed, complementary DNA oligonucleotides encoding the miRTS box for miR-7-5p (with compatible ends) into the 3′ UTR of F via PacI or of H via SpeI as described before., To generate pMV-EGFP-LmiRTS7 , the 3′ portion of the L gene, including its 3′ UTR, was amplified by high-fidelity PCR (Phusion polymerase, New England Biolabs, Frankfurt am Main, Germany) using primers that introduce artificial BamHI and SalI restriction sites for subcloning into the pUC19 vector (New England Biolabs) (forward primer, 5′-TTTTGGATCCGGCCTTGTCGAACACAGAATGG-3′; reverse primer, 5′-TTTTGTCGACTATGACCATGATTACGCCAAGC-3′). .. After subcloning, a unique SpeI restriction site was introduced by changing the L stop codon (underlined) from AC TAA T into AC TAG T (forward mutagenesis primer, 5′- CCCTGATTAAGGACTA g TTGGTTGAACTCCGGA-3′; reverse mutagenesis primer, 5′- TCCGGAGTTCAACCAA c TAGTCCTTAATCAGGG-3′).

    Article Title: MiR-891a-5p as a prognostic marker and therapeutic target for hormone receptor-positive breast cancer
    Article Snippet: .. Dual-Luciferase Reporter Assay To generate the pmirGLO-ADAM10-3'UTR reporter plasmid, the human ADAM10-3'UTR region (423 bp) encompassing the binding site of miR-891a-5pwas amplified using PCR primers (3'UTR-F: 5'-GCAGCTAGCACTAAACCCTCACAAG-3'; 3'UTR -R: 5'-TTGCGTCGACACAGAAGTACAGTGTA-3') and then cloned into the luciferase vector pmirGLO(Promega) using NheI and SalI restriction enzymes (New England Biolabs). .. T47D cells were transfected with different combinations including pmirGLO-ADAM10-3'UTR (WT) plus miR-NC; pmirGLO-ADAM10-3'UTR (WT) plus miR-891a-5p; pmirGLO-ADAM10-3'UTR (Mut) plus miR-NC; pmirGLO-ADAM10-3'UTR (Mut) plus miR-891a-5p for 48 hrs.

    Article Title: Specific disulfide cross-linking to constrict the mobile carrier domain of nonribosomal peptide synthetases
    Article Snippet: .. After amplification, the PCR product was digested with PciI and SalI restriction enzymes and ligated using T4 DNA ligase (NEB) into the vector pET28a (Novagen), which had been digested with the restriction enzymes NcoI and XhoI to create compatible sticky ends. .. The ligated vector was transformed into DH5α competent cells, and clones from the resulting colonies were verified by sequencing.

    Article Title: Profound analgesia is associated with a truncated peptide resulting from tissue specific alternative splicing of DRG CA8-204 regulated by an exon-level cis-eQTL
    Article Snippet: .. The restriction enzymes SalI and KpnI (NEB) were used for restriction digestion on PCR products and for the pCMV-N-FLAG (Takara) vector having similar restriction sites. .. CA8-204C was produced using GENEART site-directed mutagenesis system (Invitrogen Life Technologies, Carlsbad, CA) and specific primers (forward: 5’-GTTGGATTCAGTCCACGTCTTGATGTTATTT-3’, reverse: 5’-AAATAACATCAAGACGTGGACTGAATCCAAC-3’) were employed to create one nucleotide substitution at position 1416 in the CA8-204 transcript.

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    New England Biolabs sali treatment
    Bead separation from the tethered <t>DNA</t> substrate containing attB-attP by addition of <t>SalI.</t> ( A ) In the schematic representation of the 1303 bp recombination substrates (containing attB-attP or attL-attR ), the integrase cleavage sites are indicated by the short vertical arrows. The vertical line at the left and the sphere at the right indicate the tethering glass surface and the polystyrene bead, respectively. Double strand cleavage at one or both att sites will result in bead detachment from the tethered DNA. The number of molecules with detached beads following SDS treatment from three assays are tabulated. The orientation of the att sites in a substrate is indicated by the arrows. The attB-attP reactions contained integrase. The attL-attR reaction contained integrase plus gp3. ( B ) In the 1303 bp DNA substrate with head-to-tail attB-attP sites, the SalI recognition site located 241 bp away from the end attached to the glass surface. SalI digestion would release the bead along with the long DNA fragment from the short fragment that remains tethered. The BM amplitude time trace of a molecule before and after SalI addition (indicated by the short vertical arrow) are shown. The abrupt termination of the trace resulted from bead detachment. Such events from three separate experiments are tabulated. N refers to the number of DNA molecules observed.
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    Bead separation from the tethered DNA substrate containing attB-attP by addition of SalI. ( A ) In the schematic representation of the 1303 bp recombination substrates (containing attB-attP or attL-attR ), the integrase cleavage sites are indicated by the short vertical arrows. The vertical line at the left and the sphere at the right indicate the tethering glass surface and the polystyrene bead, respectively. Double strand cleavage at one or both att sites will result in bead detachment from the tethered DNA. The number of molecules with detached beads following SDS treatment from three assays are tabulated. The orientation of the att sites in a substrate is indicated by the arrows. The attB-attP reactions contained integrase. The attL-attR reaction contained integrase plus gp3. ( B ) In the 1303 bp DNA substrate with head-to-tail attB-attP sites, the SalI recognition site located 241 bp away from the end attached to the glass surface. SalI digestion would release the bead along with the long DNA fragment from the short fragment that remains tethered. The BM amplitude time trace of a molecule before and after SalI addition (indicated by the short vertical arrow) are shown. The abrupt termination of the trace resulted from bead detachment. Such events from three separate experiments are tabulated. N refers to the number of DNA molecules observed.

    Journal: Nucleic Acids Research

    Article Title: Single-molecule analysis of ϕC31 integrase-mediated site-specific recombination by tethered particle motion

    doi: 10.1093/nar/gkw861

    Figure Lengend Snippet: Bead separation from the tethered DNA substrate containing attB-attP by addition of SalI. ( A ) In the schematic representation of the 1303 bp recombination substrates (containing attB-attP or attL-attR ), the integrase cleavage sites are indicated by the short vertical arrows. The vertical line at the left and the sphere at the right indicate the tethering glass surface and the polystyrene bead, respectively. Double strand cleavage at one or both att sites will result in bead detachment from the tethered DNA. The number of molecules with detached beads following SDS treatment from three assays are tabulated. The orientation of the att sites in a substrate is indicated by the arrows. The attB-attP reactions contained integrase. The attL-attR reaction contained integrase plus gp3. ( B ) In the 1303 bp DNA substrate with head-to-tail attB-attP sites, the SalI recognition site located 241 bp away from the end attached to the glass surface. SalI digestion would release the bead along with the long DNA fragment from the short fragment that remains tethered. The BM amplitude time trace of a molecule before and after SalI addition (indicated by the short vertical arrow) are shown. The abrupt termination of the trace resulted from bead detachment. Such events from three separate experiments are tabulated. N refers to the number of DNA molecules observed.

    Article Snippet: SalI treatment of tethered DNA molecules The DNA molecules were tethered to the glass surface in the reaction chamber containing the SalI digestion buffer (from New England BioLabs).

    Techniques: