bstxi  (New England Biolabs)


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    Name:
    BstXI
    Description:
    BstXI 5 000 units
    Catalog Number:
    r0113l
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    Structured Review

    New England Biolabs bstxi
    BstXI
    BstXI 5 000 units
    https://www.bioz.com/result/bstxi/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bstxi - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †"

    Article Title: Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †

    Journal: Biochemistry

    doi: 10.1021/bi902169q

    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    Figure Legend Snippet: A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Techniques Used: Plasmid Preparation, Ligation, Blocking Assay, Labeling

    2) Product Images from "Sirtuin 1 Is a Key Regulator of the Interleukin-12 p70/Interleukin-23 Balance in Human Dendritic Cells *"

    Article Title: Sirtuin 1 Is a Key Regulator of the Interleukin-12 p70/Interleukin-23 Balance in Human Dendritic Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.391839

    Accessibility of Nuc-1 and Nuc-2 of the il12a promoter. Accessibility was assayed by CHART-PCR. Nuclei were digested with 50 units BstXI or 50 units PshAI for 1 h at 37 °C, and genomic DNA was used to perform SYBR Green quantitative PCR. Amplification with primers for the Nuc-1-encompassing PshAI site is sensitive to remodeling of Nuc-1, and amplification with primers for Nuc-2 encompassing BstXI is sensitive to remodeling of Nuc-2. Results are expressed as a percentage of the undigested sample for each cell treatment as described under “Experimental Procedures.” Results represent the mean ± S.D. of five experiments with triplicate samples. *, p
    Figure Legend Snippet: Accessibility of Nuc-1 and Nuc-2 of the il12a promoter. Accessibility was assayed by CHART-PCR. Nuclei were digested with 50 units BstXI or 50 units PshAI for 1 h at 37 °C, and genomic DNA was used to perform SYBR Green quantitative PCR. Amplification with primers for the Nuc-1-encompassing PshAI site is sensitive to remodeling of Nuc-1, and amplification with primers for Nuc-2 encompassing BstXI is sensitive to remodeling of Nuc-2. Results are expressed as a percentage of the undigested sample for each cell treatment as described under “Experimental Procedures.” Results represent the mean ± S.D. of five experiments with triplicate samples. *, p

    Techniques Used: Polymerase Chain Reaction, SYBR Green Assay, Real-time Polymerase Chain Reaction, Amplification

    Related Articles

    Polymerase Chain Reaction:

    Article Title: A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens
    Article Snippet: The iProof ™ high fidelity Taq DNA polymerase (Bio-Rad, Hercules, CA) PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). .. The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA). .. The destination binary plasmid pEarleyGate101 [ ] was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH) and digested with Bbv CI and Bst XI to remove the ccdB gene.

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging
    Article Snippet: Generation of Split GFP Expressing Stable Cells To allow detection of CRISPR edited cells we included a split GFP tag developed in the Bo Huang lab . .. The sfGFP1-10 barrel was synthesised and cloned into a lentiviral vector (Vectorbuilder), and further subcloned into a CMV driven vector (pLenti CMV V5-LUC Blast (w567-1) was a gift from Eric Campeau (Addgene plasmid #21474 ; http://n2t.net/addgene:21474 ; RRID:Addgene_21474 )), briefly FLuc was removed from the vector by digesting with BstXI. sfGFP1-10 was PCR amplified with addition of a signal peptide to target expression to the endoplasmic reticulum (ER), using primers in , and assembled using a Gibson Assembly master mix (NEB). .. 5 μg pLV-ERsfGfp1-10 was then transfected into 293T cells, along with 2.5 μg VSVG, 2.5 μg pRSV-Rev and 2.5 μg pMDLg/pRRE, using a 3:1 ratio of PEI:DNA, to generate lentivirus, medium was collected 24-48hours post transfection, filtered through a 0.45 μm filter and added to NIH3T3 cells with 8 μg/mL polybrene.

    Article Title: A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns
    Article Snippet: Methylation-sensitive Endonuclease Digestion Genomic DNA was isolated from 7-dpa cotton ovules harvested at different times of the year. .. Aliquots (1 µg) were digested by incubation with 1 µl Bsl I (10,000 U/ml) in NEBuffer 3 at 55°C for 16 h, with 1 µl HinF I (10,000 U/ml) in NEBuffer 4 at 37°C for 16 h, or with 1 µl BstX I (10,000 U/ml) in NEBuffer 3 at 37°C for 16 h. One twentieth of the digested DNA sample was used as the template for each PCR analysis. .. Southern Blots To detect cleavage of methylation-sensitive sites, 20 µg genomic DNA was digested thoroughly (using 20 times the amount of enzyme specified in the previous section using methylation-sensitive Bsl I, HinF I, or BstX I, loaded onto electrophoretic gels, and blotted prior to hybridization and detection using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) with probe sequences specified in each respective figure panel.

    Purification:

    Article Title: A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens
    Article Snippet: The iProof ™ high fidelity Taq DNA polymerase (Bio-Rad, Hercules, CA) PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). .. The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA). .. The destination binary plasmid pEarleyGate101 [ ] was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH) and digested with Bbv CI and Bst XI to remove the ccdB gene.

    Gel Purification:

    Article Title: A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens
    Article Snippet: The iProof ™ high fidelity Taq DNA polymerase (Bio-Rad, Hercules, CA) PCR program consisted of 1 cycle at 98°C (2 min), followed by 30 cycles at 98°C (30 s), 55°C (45 s), and 72°C (1 min), and finished with a 1 cycle extension at 72°C (7 min). .. The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA). .. The destination binary plasmid pEarleyGate101 [ ] was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus, OH) and digested with Bbv CI and Bst XI to remove the ccdB gene.

    Oligonucleotide Synthesis:

    Article Title: Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †
    Article Snippet: We found that ICLs that covalently block the hydrogen bond face of the base display significantly lower repair efficiencies than ICLs that do not interfere with the hydrogen bond face. .. Protected deoxyribonucleoside 3′-O-phosphoramidites and oligonucleotide synthesis reagents were obtained from Glen Research, Inc. Polynucleotide kinase, T4 DNA ligase, E. coli DNA polymerase I (Klenow fragment), EcoRI, and BstXI were obtained from New England Biolabs, Inc. Van91I was obtained from Fermantas, Inc. .. All oligonucleotides were synthesized on an ABI DNA synthesizer (model 3400) and purified using high performance liquid chromatography (HPLC) on a Varian instrument using a 0.4 × 25 cm Dionex strong anion exchange (SAX) column.

    Clone Assay:

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging
    Article Snippet: Generation of Split GFP Expressing Stable Cells To allow detection of CRISPR edited cells we included a split GFP tag developed in the Bo Huang lab . .. The sfGFP1-10 barrel was synthesised and cloned into a lentiviral vector (Vectorbuilder), and further subcloned into a CMV driven vector (pLenti CMV V5-LUC Blast (w567-1) was a gift from Eric Campeau (Addgene plasmid #21474 ; http://n2t.net/addgene:21474 ; RRID:Addgene_21474 )), briefly FLuc was removed from the vector by digesting with BstXI. sfGFP1-10 was PCR amplified with addition of a signal peptide to target expression to the endoplasmic reticulum (ER), using primers in , and assembled using a Gibson Assembly master mix (NEB). .. 5 μg pLV-ERsfGfp1-10 was then transfected into 293T cells, along with 2.5 μg VSVG, 2.5 μg pRSV-Rev and 2.5 μg pMDLg/pRRE, using a 3:1 ratio of PEI:DNA, to generate lentivirus, medium was collected 24-48hours post transfection, filtered through a 0.45 μm filter and added to NIH3T3 cells with 8 μg/mL polybrene.

    Plasmid Preparation:

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging
    Article Snippet: Generation of Split GFP Expressing Stable Cells To allow detection of CRISPR edited cells we included a split GFP tag developed in the Bo Huang lab . .. The sfGFP1-10 barrel was synthesised and cloned into a lentiviral vector (Vectorbuilder), and further subcloned into a CMV driven vector (pLenti CMV V5-LUC Blast (w567-1) was a gift from Eric Campeau (Addgene plasmid #21474 ; http://n2t.net/addgene:21474 ; RRID:Addgene_21474 )), briefly FLuc was removed from the vector by digesting with BstXI. sfGFP1-10 was PCR amplified with addition of a signal peptide to target expression to the endoplasmic reticulum (ER), using primers in , and assembled using a Gibson Assembly master mix (NEB). .. 5 μg pLV-ERsfGfp1-10 was then transfected into 293T cells, along with 2.5 μg VSVG, 2.5 μg pRSV-Rev and 2.5 μg pMDLg/pRRE, using a 3:1 ratio of PEI:DNA, to generate lentivirus, medium was collected 24-48hours post transfection, filtered through a 0.45 μm filter and added to NIH3T3 cells with 8 μg/mL polybrene.

    Amplification:

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging
    Article Snippet: Generation of Split GFP Expressing Stable Cells To allow detection of CRISPR edited cells we included a split GFP tag developed in the Bo Huang lab . .. The sfGFP1-10 barrel was synthesised and cloned into a lentiviral vector (Vectorbuilder), and further subcloned into a CMV driven vector (pLenti CMV V5-LUC Blast (w567-1) was a gift from Eric Campeau (Addgene plasmid #21474 ; http://n2t.net/addgene:21474 ; RRID:Addgene_21474 )), briefly FLuc was removed from the vector by digesting with BstXI. sfGFP1-10 was PCR amplified with addition of a signal peptide to target expression to the endoplasmic reticulum (ER), using primers in , and assembled using a Gibson Assembly master mix (NEB). .. 5 μg pLV-ERsfGfp1-10 was then transfected into 293T cells, along with 2.5 μg VSVG, 2.5 μg pRSV-Rev and 2.5 μg pMDLg/pRRE, using a 3:1 ratio of PEI:DNA, to generate lentivirus, medium was collected 24-48hours post transfection, filtered through a 0.45 μm filter and added to NIH3T3 cells with 8 μg/mL polybrene.

    Expressing:

    Article Title: Dynamic protein quantitation (DyProQ) of procollagen-I by CRISPR-Cas9 NanoLuciferase tagging
    Article Snippet: Generation of Split GFP Expressing Stable Cells To allow detection of CRISPR edited cells we included a split GFP tag developed in the Bo Huang lab . .. The sfGFP1-10 barrel was synthesised and cloned into a lentiviral vector (Vectorbuilder), and further subcloned into a CMV driven vector (pLenti CMV V5-LUC Blast (w567-1) was a gift from Eric Campeau (Addgene plasmid #21474 ; http://n2t.net/addgene:21474 ; RRID:Addgene_21474 )), briefly FLuc was removed from the vector by digesting with BstXI. sfGFP1-10 was PCR amplified with addition of a signal peptide to target expression to the endoplasmic reticulum (ER), using primers in , and assembled using a Gibson Assembly master mix (NEB). .. 5 μg pLV-ERsfGfp1-10 was then transfected into 293T cells, along with 2.5 μg VSVG, 2.5 μg pRSV-Rev and 2.5 μg pMDLg/pRRE, using a 3:1 ratio of PEI:DNA, to generate lentivirus, medium was collected 24-48hours post transfection, filtered through a 0.45 μm filter and added to NIH3T3 cells with 8 μg/mL polybrene.

    Incubation:

    Article Title: Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements
    Article Snippet: φX174 virion DNA was annealed to oligonucleotide P-6 containing a branch point adjacent to a Bst XI recognition sequence and extended by 3′-5′ exonuclease-negative Pfu DNA polymerase (Stratagene) for 18 cycles of 55°C for 1 min and 65°C for 25 min. An aliquot was digested by Hae III and analyzed by agarose gel electrophoresis to verify conversion to duplex DNA. .. Various concentrations of FEN and 500 ng of branched duplex φX174 were incubated in 50 mM KCl–10 mM Tris-HCl (pH 8.0)–2.5 mM MgCl2 –1 mM DTT–10 μg of bovine serum albumin (BSA) per ml at 55°C for 1 h and digested with 1 U each of Bst XI and Bsi EI (NEB) at 55°C for 1 h. Products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized by fluorography. .. The oligonucleotide FS was phosphorylated with [γ-32 P]ATP and purified by phenol extraction and ethanol precipitation.

    Article Title: A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns
    Article Snippet: Methylation-sensitive Endonuclease Digestion Genomic DNA was isolated from 7-dpa cotton ovules harvested at different times of the year. .. Aliquots (1 µg) were digested by incubation with 1 µl Bsl I (10,000 U/ml) in NEBuffer 3 at 55°C for 16 h, with 1 µl HinF I (10,000 U/ml) in NEBuffer 4 at 37°C for 16 h, or with 1 µl BstX I (10,000 U/ml) in NEBuffer 3 at 37°C for 16 h. One twentieth of the digested DNA sample was used as the template for each PCR analysis. .. Southern Blots To detect cleavage of methylation-sensitive sites, 20 µg genomic DNA was digested thoroughly (using 20 times the amount of enzyme specified in the previous section using methylation-sensitive Bsl I, HinF I, or BstX I, loaded onto electrophoretic gels, and blotted prior to hybridization and detection using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) with probe sequences specified in each respective figure panel.

    Agarose Gel Electrophoresis:

    Article Title: Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements
    Article Snippet: φX174 virion DNA was annealed to oligonucleotide P-6 containing a branch point adjacent to a Bst XI recognition sequence and extended by 3′-5′ exonuclease-negative Pfu DNA polymerase (Stratagene) for 18 cycles of 55°C for 1 min and 65°C for 25 min. An aliquot was digested by Hae III and analyzed by agarose gel electrophoresis to verify conversion to duplex DNA. .. Various concentrations of FEN and 500 ng of branched duplex φX174 were incubated in 50 mM KCl–10 mM Tris-HCl (pH 8.0)–2.5 mM MgCl2 –1 mM DTT–10 μg of bovine serum albumin (BSA) per ml at 55°C for 1 h and digested with 1 U each of Bst XI and Bsi EI (NEB) at 55°C for 1 h. Products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized by fluorography. .. The oligonucleotide FS was phosphorylated with [γ-32 P]ATP and purified by phenol extraction and ethanol precipitation.

    Staining:

    Article Title: Methanococcus jannaschii Flap Endonuclease: Expression, Purification, and Substrate Requirements
    Article Snippet: φX174 virion DNA was annealed to oligonucleotide P-6 containing a branch point adjacent to a Bst XI recognition sequence and extended by 3′-5′ exonuclease-negative Pfu DNA polymerase (Stratagene) for 18 cycles of 55°C for 1 min and 65°C for 25 min. An aliquot was digested by Hae III and analyzed by agarose gel electrophoresis to verify conversion to duplex DNA. .. Various concentrations of FEN and 500 ng of branched duplex φX174 were incubated in 50 mM KCl–10 mM Tris-HCl (pH 8.0)–2.5 mM MgCl2 –1 mM DTT–10 μg of bovine serum albumin (BSA) per ml at 55°C for 1 h and digested with 1 U each of Bst XI and Bsi EI (NEB) at 55°C for 1 h. Products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized by fluorography. .. The oligonucleotide FS was phosphorylated with [γ-32 P]ATP and purified by phenol extraction and ethanol precipitation.

    Recombinant:

    Article Title: Sirtuin 1 Is a Key Regulator of the Interleukin-12 p70/Interleukin-23 Balance in Human Dendritic Cells *
    Article Snippet: 3-Aminopyridine adenine dinucleotide (AAD), a NAD+ analog that increases the cellular concentration of NAD+ by inhibiting dehydrogenases , was synthesized from nicotinic acid adenine dinucleotide and sodium azide as described ( ). .. Recombinant histone H3.3, BstXI, and PshAI were from New England BioLabs (Ipswich, MA). p300, pCAF, and SIRT1 recombinant proteins were from Active Motif (Carlsbad, CA). .. SRT1720, a selective activator of SIRT1, was from Cayman Chemical (Ann Arbor, MI).

    Concentration Assay:

    Article Title: Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model
    Article Snippet: A consensus sequence for each of the cloned fragments was determined, and when necessary (i.e., pTGEV A, pTGEV B1, pTGEV C, and pTGEV F), a consensus clone was assembled using restriction enzymes and standard recombinant DNA techniques to remove unwanted amino acid changes associated with reverse transcription or naturally occurring quasispecies variation. .. Each of the plasmids was grown to high concentration, isolated, and digested or double-digested with Bgl I, Bst XI, or Not I according to the manufacturer's direction (NEB) (Fig. A). ..

    Isolation:

    Article Title: Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model
    Article Snippet: A consensus sequence for each of the cloned fragments was determined, and when necessary (i.e., pTGEV A, pTGEV B1, pTGEV C, and pTGEV F), a consensus clone was assembled using restriction enzymes and standard recombinant DNA techniques to remove unwanted amino acid changes associated with reverse transcription or naturally occurring quasispecies variation. .. Each of the plasmids was grown to high concentration, isolated, and digested or double-digested with Bgl I, Bst XI, or Not I according to the manufacturer's direction (NEB) (Fig. A). ..

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    New England Biolabs bstxi
    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and <t>BstXI</t> to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli <t>DNA</t> polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    Bstxi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bstxi/product/New England Biolabs
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    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Journal: Biochemistry

    Article Title: Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †

    doi: 10.1021/bi902169q

    Figure Lengend Snippet: A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Article Snippet: Protected deoxyribonucleoside 3′-O-phosphoramidites and oligonucleotide synthesis reagents were obtained from Glen Research, Inc. Polynucleotide kinase, T4 DNA ligase, E. coli DNA polymerase I (Klenow fragment), EcoRI, and BstXI were obtained from New England Biolabs, Inc. Van91I was obtained from Fermantas, Inc.

    Techniques: Plasmid Preparation, Ligation, Blocking Assay, Labeling

    Marker mutations are present in virus derived from the infectious construct. Cultures of ST cells were infected with wild-type (WT) TGEV or plaque-purified icTGEV isolates derived from the infectious construct. Intracellular RNA was isolated, and RT-PCR was performed using primer pairs that asymmetrically flank each of the unique Bgl I- Bst XI junctions inserted into the infectious construct. (A) Wild-type TGEV. (B) icTGEV-1 (passage 1). (C) icTGEV-3 (passage 3). In panel C, a larger ∼1.6-kb wild-type TGEV amplicon spanning the B1-B2 junction was also treated with Bst ]). Arrows indicate cleaved DNA intermediates.

    Journal: Journal of Virology

    Article Title: Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

    doi:

    Figure Lengend Snippet: Marker mutations are present in virus derived from the infectious construct. Cultures of ST cells were infected with wild-type (WT) TGEV or plaque-purified icTGEV isolates derived from the infectious construct. Intracellular RNA was isolated, and RT-PCR was performed using primer pairs that asymmetrically flank each of the unique Bgl I- Bst XI junctions inserted into the infectious construct. (A) Wild-type TGEV. (B) icTGEV-1 (passage 1). (C) icTGEV-3 (passage 3). In panel C, a larger ∼1.6-kb wild-type TGEV amplicon spanning the B1-B2 junction was also treated with Bst ]). Arrows indicate cleaved DNA intermediates.

    Article Snippet: Each of the plasmids was grown to high concentration, isolated, and digested or double-digested with Bgl I, Bst XI, or Not I according to the manufacturer's direction (NEB) (Fig. A).

    Techniques: Marker, Derivative Assay, Construct, Infection, Purification, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification

    Assembly of the TGEV full-length construct. (A) The various TGEV plasmid DNAs were digested with Bgl I, Bst XI, or Not I, and the appropriate-sized products were isolated from agarose gels as described in the text. The TGEV A and B1 fragments, TGEV B2 and C3 fragments, or TGEV DE-1 and F fragments were ligated at 16°C overnight in separate reactions. Appropriate-sized products were isolated from agarose gels. (B) A+B1. (C) B2+C. (D) DE-1+F. Following purification from agarose gels, the purified products are shown in panel A as well.

    Journal: Journal of Virology

    Article Title: Strategy for Systematic Assembly of Large RNA and DNA Genomes: Transmissible Gastroenteritis Virus Model

    doi:

    Figure Lengend Snippet: Assembly of the TGEV full-length construct. (A) The various TGEV plasmid DNAs were digested with Bgl I, Bst XI, or Not I, and the appropriate-sized products were isolated from agarose gels as described in the text. The TGEV A and B1 fragments, TGEV B2 and C3 fragments, or TGEV DE-1 and F fragments were ligated at 16°C overnight in separate reactions. Appropriate-sized products were isolated from agarose gels. (B) A+B1. (C) B2+C. (D) DE-1+F. Following purification from agarose gels, the purified products are shown in panel A as well.

    Article Snippet: Each of the plasmids was grown to high concentration, isolated, and digested or double-digested with Bgl I, Bst XI, or Not I according to the manufacturer's direction (NEB) (Fig. A).

    Techniques: Construct, Plasmid Preparation, Isolation, Purification

    Schematic cloning of a gene into a binary expression vector and selection in Agrobacterium tumefaciens . (A) Key features of pEG101-SacB/R. LB: T-DNA left border; RB: T-DNA right border; Cm R : chloramphenicol resistance gene; attL1 and attL2: recognition sites of the LR clonase; ccdB : the Invitrogen ccdB gene cassette frame B; SacB/SacR : Levansucrase genes along with their native promoter; Bbv CI and Bst XI: restriction enzyme sites for replacing the ccdB gene fragment with the SacB/SacR gene cassette. (B). Compared to the regular LR cloning procedure, the new protocol is more convenient and time-saving.

    Journal: Plant Methods

    Article Title: A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

    doi: 10.1186/1746-4811-7-42

    Figure Lengend Snippet: Schematic cloning of a gene into a binary expression vector and selection in Agrobacterium tumefaciens . (A) Key features of pEG101-SacB/R. LB: T-DNA left border; RB: T-DNA right border; Cm R : chloramphenicol resistance gene; attL1 and attL2: recognition sites of the LR clonase; ccdB : the Invitrogen ccdB gene cassette frame B; SacB/SacR : Levansucrase genes along with their native promoter; Bbv CI and Bst XI: restriction enzyme sites for replacing the ccdB gene fragment with the SacB/SacR gene cassette. (B). Compared to the regular LR cloning procedure, the new protocol is more convenient and time-saving.

    Article Snippet: The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA).

    Techniques: Clone Assay, Expressing, Plasmid Preparation, Selection

    The destination vector pEG101-SacB/R can be stably propagated in the E. coli strain DH5α . Restriction digestion ( Bst XI and Bbv CI) of plasmid DNAs, isolated from five repeated overnight cultures of E. coli carrying the pEG101-SacB/R destination vector, showed identical restriction patterns. M: 1 kb marker. Lanes 1 through 5 were digested plasmid DNAs of five repeated subcultures.

    Journal: Plant Methods

    Article Title: A novel Gateway®-compatible binary vector allows direct selection of recombinant clones in Agrobacterium tumefaciens

    doi: 10.1186/1746-4811-7-42

    Figure Lengend Snippet: The destination vector pEG101-SacB/R can be stably propagated in the E. coli strain DH5α . Restriction digestion ( Bst XI and Bbv CI) of plasmid DNAs, isolated from five repeated overnight cultures of E. coli carrying the pEG101-SacB/R destination vector, showed identical restriction patterns. M: 1 kb marker. Lanes 1 through 5 were digested plasmid DNAs of five repeated subcultures.

    Article Snippet: The PCR products were digested with Bbv CI and Bst XI (New England BioLabs Inc, Ipswich, MA), and gel purified using an AccuPrep™ Gel Purification Kit (Bioneer, Alameda, CA).

    Techniques: Plasmid Preparation, Stable Transfection, Isolation, Marker

    Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions over one year. (A) Methylation-sensitive endonuclease digested PCR amplification of ERF6 upstream region. Top: schematic diagram of the identification of a methylation-sensitive BstX I digenstion site (CCANNNNNNTGG) at −275 bp of the ERF6 promoter. The bold C indicates a CHH site with annual methylation pattern change, corresponding to the cytosine labelled with red triangles in Figure 3A . Bottom: PCR amplification using genomic DNA with (+) or without (−) BstX I digestion. (B) Southern blot of genomic DNA harvested at different times of the year, first digested by a methylation non-sensitive endonuclease Mbo II (TCTTC) to obtain a full length fragment of 605 bp from −621 to −15 of ERF6 upstream regions, then digested thoroughly with BstX I, and probed with the fragment from −263 to −21 nt. The signal intensities of the band of BstX I-cleaved 244 bp changed at different time-of-year (see Table S8 ), indicating the methlytion levels of this CHH site were different, consistent with the bisulfite sequencing data in Figure 3A and methylation-sensitive endonuclease digested PCR results in Figure 4A . The same methylation-sensitive endonuclease digested PCR experiments were performed for the upstream regions of SUR4 (C) and KCS13 (E), except the methylation-sensitive endonucleases used were HinF I and Bsl I, respectively. Further, the same methylation-sensitive endonuclease digested Southern experiments were performed for the upstream regions of SUR4 (D) and KCS13 (F), except the genomic DNA were first digested by Bcl I (TGATCA) and NSi I (ATGCAT), then digested by methylation-sensitive endonucleases HinF I (GANTC) and Bsl I (CCNNNNNNNGG), respectively. The signal intensities of HinF I- and Bsl I-cleaved 330 bp and 837 bp changed similarly (see Table S8 ).

    Journal: PLoS ONE

    Article Title: A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

    doi: 10.1371/journal.pone.0060547

    Figure Lengend Snippet: Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions over one year. (A) Methylation-sensitive endonuclease digested PCR amplification of ERF6 upstream region. Top: schematic diagram of the identification of a methylation-sensitive BstX I digenstion site (CCANNNNNNTGG) at −275 bp of the ERF6 promoter. The bold C indicates a CHH site with annual methylation pattern change, corresponding to the cytosine labelled with red triangles in Figure 3A . Bottom: PCR amplification using genomic DNA with (+) or without (−) BstX I digestion. (B) Southern blot of genomic DNA harvested at different times of the year, first digested by a methylation non-sensitive endonuclease Mbo II (TCTTC) to obtain a full length fragment of 605 bp from −621 to −15 of ERF6 upstream regions, then digested thoroughly with BstX I, and probed with the fragment from −263 to −21 nt. The signal intensities of the band of BstX I-cleaved 244 bp changed at different time-of-year (see Table S8 ), indicating the methlytion levels of this CHH site were different, consistent with the bisulfite sequencing data in Figure 3A and methylation-sensitive endonuclease digested PCR results in Figure 4A . The same methylation-sensitive endonuclease digested PCR experiments were performed for the upstream regions of SUR4 (C) and KCS13 (E), except the methylation-sensitive endonucleases used were HinF I and Bsl I, respectively. Further, the same methylation-sensitive endonuclease digested Southern experiments were performed for the upstream regions of SUR4 (D) and KCS13 (F), except the genomic DNA were first digested by Bcl I (TGATCA) and NSi I (ATGCAT), then digested by methylation-sensitive endonucleases HinF I (GANTC) and Bsl I (CCNNNNNNNGG), respectively. The signal intensities of HinF I- and Bsl I-cleaved 330 bp and 837 bp changed similarly (see Table S8 ).

    Article Snippet: Aliquots (1 µg) were digested by incubation with 1 µl Bsl I (10,000 U/ml) in NEBuffer 3 at 55°C for 16 h, with 1 µl HinF I (10,000 U/ml) in NEBuffer 4 at 37°C for 16 h, or with 1 µl BstX I (10,000 U/ml) in NEBuffer 3 at 37°C for 16 h. One twentieth of the digested DNA sample was used as the template for each PCR analysis.

    Techniques: Methylation, Polymerase Chain Reaction, Amplification, Southern Blot, Methylation Sequencing

    Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions in ROS1 RNAi lines. (A) Analysis of relative ERF6 transcription in ovules from ROS1 RNAi lines by qRT-PCR. The level of ERF6 transcripts in ovules from the empty vector line (V) was arbitrarily defined as 1. (B) Southern blot analysis of genomic DNA prepared from ROS1 RNAi lines digested thoroughly with BstX I. See detailed information in Figure 4 legend. Similar qRT-PCR experiments were performed for SUR4 (C) and KCS13 (E) transcriptions, as well as similar Southern experiments for SUR4 (D) and KCS13 (F), respectively. Note the reduced intensities of the BstX I-, HinF I- and Bsl I-cleaved bands in all three RNAi lines compared to the vector line.

    Journal: PLoS ONE

    Article Title: A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

    doi: 10.1371/journal.pone.0060547

    Figure Lengend Snippet: Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions in ROS1 RNAi lines. (A) Analysis of relative ERF6 transcription in ovules from ROS1 RNAi lines by qRT-PCR. The level of ERF6 transcripts in ovules from the empty vector line (V) was arbitrarily defined as 1. (B) Southern blot analysis of genomic DNA prepared from ROS1 RNAi lines digested thoroughly with BstX I. See detailed information in Figure 4 legend. Similar qRT-PCR experiments were performed for SUR4 (C) and KCS13 (E) transcriptions, as well as similar Southern experiments for SUR4 (D) and KCS13 (F), respectively. Note the reduced intensities of the BstX I-, HinF I- and Bsl I-cleaved bands in all three RNAi lines compared to the vector line.

    Article Snippet: Aliquots (1 µg) were digested by incubation with 1 µl Bsl I (10,000 U/ml) in NEBuffer 3 at 55°C for 16 h, with 1 µl HinF I (10,000 U/ml) in NEBuffer 4 at 37°C for 16 h, or with 1 µl BstX I (10,000 U/ml) in NEBuffer 3 at 37°C for 16 h. One twentieth of the digested DNA sample was used as the template for each PCR analysis.

    Techniques: Methylation, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Southern Blot