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  • 99
    New England Biolabs bamhi
    Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a <t>DNA</t> fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, <t>BamHI,</t> which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamhi
    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With <t>BamHI</t> and <t>SalI</t> RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .
    Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14082 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore bamhi
    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The <t>PstI-BamHI</t> fragment of <t>pVP9</t> is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids
    Bamhi, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6675 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa bamhi
    <t>Integron</t> comparisons based on <t>BamHI</t> digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696
    Bamhi, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 6140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs bamhi hf
    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and <t>BamHI-HF,</t> into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) <t>DNA</t> from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.
    Bamhi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamh i
    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the <t>BamH</t> I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.
    Bamh I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene bamhi site
    Radioactive RT-PCR assay for detecting the use of alternative 3′ splice sites. The assay was conducted with exon-specific primers I4B_right (radiolabeled) and <t>5p_left_BamHI</t> ( Table 1 ) with wild-type (WT) and d3ps mutant constructs spliced in vitro in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 and 100 mM MgCl 2 at 45°C. While no alternative splicing was detected for the WT B.c .I4 intron, the d3ps mutant could use a 3′ splice site at position +2 downstream of the mutated wild-type site. Quantification of the bands using a phosphorimager is shown, expressed as percentage of total radioactivity. The intron-3′ exon splice junction is shown on the right, with mutated nucleotides boxed.
    Bamhi Site, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 2647 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher fastdigest bamhi
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Fastdigest Bamhi, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bamhi hindiii sites
    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total <t>DNA</t> (7 µg) was digested with <t>BamHI</t> which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.
    Bamhi Hindiii Sites, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 225 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).

    Journal: mBio

    Article Title: Genome-Scale Identification of Resistance Functions in Pseudomonas aeruginosa Using Tn-seq

    doi: 10.1128/mBio.00315-10

    Figure Lengend Snippet: Tn-seq circle method. The steps used to amplify and sequence transposon insertion junctions are illustrated, beginning with a DNA fragment carrying a transposon insertion (top). First, total DNA from a mutant pool is sheared and end repaired, and one Illumina adaptor (A2) is ligated to all free ends (step 1). The sample is then digested with a restriction enzyme that cuts near one transposon end (in this work, BamHI, which cuts 114 bp from the transposon’s left end) (step 2). Following a size selection step, single-strand fragments which include the transposon end are circularized by templated ligation (step 3). Oligo, oligonucleotide. Fragments which have not circularized (representing most of the DNA in the sample) are degraded in a subsequent exonuclease step (step 4). The transposon-genome junctions from the circularized fragments are then amplified by quantitative PCR in a step in which the second required Illumina adaptor (A1) is introduced (step 5). The products are sequenced on an Illumina flow cell using a sequencing primer corresponding to the transposon end (Seq), and each sequence read is then mapped to the genome (step 6).

    Article Snippet: The DNA was then digested overnight at 37°C with BamHI (NEB).

    Techniques: Sequencing, Mutagenesis, Selection, Ligation, Amplification, Real-time Polymerase Chain Reaction

    TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Journal: Virology Journal

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein

    doi: 10.1186/1743-422X-9-279

    Figure Lengend Snippet: TMV-CP DNA fragments (1% agarose gel). ( A ) The whole TMV-CP fragments with BamH I and Xho I restriction enzyme cutting sites which have been cloned in PGEX-6P-1. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a positive control with DNA template. Lane 3 is a negative control without DNA template. ( B ) The whole TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. As shown in lane 1, amplified PCR product ran at approximately 500 bp compared with the DNA marker (lane M). Lane 2 is a negative control without DNA template. Lane 3 is a positive control with DNA template. ( C ) The truncation of four amino acids from the C-terminus of TMV-CP fragments with Nde I and Xho I restriction enzyme cutting sites that have been cloned in pET28a. Lane 1 is a negative control without DNA template, whereas lane 2 is a positive control with DNA template. Lane 3 is the amplified PCR product that ran at approximately 500 bp compared with the DNA marker (lane M).

    Article Snippet: Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ).

    Techniques: Agarose Gel Electrophoresis, Clone Assay, Amplification, Polymerase Chain Reaction, Marker, Positive Control, Negative Control

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

    Journal: Bioengineered

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae

    doi: 10.4161/bioe.29167

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

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

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

    Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

    Journal: PLoS ONE

    Article Title: Mechanism of Human Papillomavirus Binding to Human Spermatozoa and Fertilizing Ability of Infected Spermatozoa

    doi: 10.1371/journal.pone.0015036

    Figure Lengend Snippet: Sperm transfected with E6/E7 plasmid penetrate oocytes. a . Scheme of the recombinant plasmid pIRES2-AcGFP1-E6E7. E6/E7 genes have been amplified (1032 bp) from plasmid p1321 HPV-16 E6/E7 by PCR and subcloned to plasmid pIRES2-AcGFP1 between SalI and BamHI restriction sites. b . PCR for HPV E6/E7 genes from transfected sperm. Lane M: DNA marker (100 bp); 1: sperm transfected with recombinant E6/E7 plasmid; 2: negative control (no template); 3: sperm transfected only with Lipofectamine 2000; 4: positive control (pIRES2-AcGFP1-E6E7 plasmid). c . Mean number of human sperm penetrated per hamster oocyte in control and sperm transfected with HPV-16 E6/E7 plasmid. d . Hamster oocytes penetrated by control sperm and sperm transfected with HPV16 E6/E7 plasmid in bright field (BF, upper panel) and fluorescence (FL, lower panel) using SYBR green DNA stain.

    Article Snippet: The PCR mixture consisted of 5 µL Expand high fidelity buffer with MgCl2 , 1 µL of PCR grade nucleotide mix (10×), 20 pmol of each primer with SalI and BamHI restriction sites (New England Biolabs, Ipswich, MA) including forward: 5′-GTGCGCGTCGACGTGATGCACCAAAAGAGAACTG-3′ and reverse: 5′-GTGCGCGGATCCGTGTGGTTTCTGAGAACAGATG-3′ , 0,75 µL Expand high fidelity enzyme mix (Expand High Fidelity PCR System dNTPack, Roche Applied Science, Mannheim, Germany) and sterile H2 O in a final volume of 50 μL.

    Techniques: Transfection, Plasmid Preparation, Recombinant, Amplification, Polymerase Chain Reaction, Marker, Negative Control, Positive Control, Fluorescence, SYBR Green Assay, Staining

    A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Journal: Jundishapur Journal of Microbiology

    Article Title: Designing, Construction and Expression of a Recombinant Fusion Protein Comprising the Hepatitis E Virus ORF2 and Rotavirus NSP4 in the Baculovirus Expression System

    doi: 10.5812/jjm.40303

    Figure Lengend Snippet: A: pFastBac1 and pBlueScript-ORF2-NSP4 Genes is Double Digested With BamHI and SalI RESTRICTION ENZYMES A: The linearized pFastBac1 and ORF2-NSP4 respectively appeared at 4775 and 2000 bp. Lane 1: pFastBac1 is double digested with BamHI/SalI ; Lane 2: pBlueScript-ORF2-NSP4 is double digested with BamHI/SalI.B : ORF2-NSP4 gene was cloned in pFastBac1. Lane 1 - 2: pFastBac1-ORF2-NSP4; and Lane3 - 4: pFastBac1. C: the position site of ORF2-NSP4 was confirmed by single and double digestion and the length of subcloned genes after single digestion was 6775 bp while after double digestion was 4775 and 2000 bp. Lane 1 - 2: pFastBac1-ORF2-NSP4 single digestion BamHI ; Lane 3 - 4: pFastBac1-ORF2-NSP4 single digestion SalI; and Lane 5-6: pFastBac1-ORF2-NSP4 double digestion BamHI/SalI .

    Article Snippet: The pBlue Script II containing fusion truncated ORF2-NSP4 was transformed in E.coli DH5α, cultured in LB agar containing ampicillin (50 mg/L), then plasmid was extracted and double digested by BamHI and SalI restriction enzymes (Thermo Scientific, USA).

    Techniques: Clone Assay

    Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Journal: Molecular and Cellular Biology

    Article Title: Differential Contributions of Mammalian Rad54 Paralogs to Recombination, DNA Damage Repair, and Meiosis †

    doi: 10.1128/MCB.26.3.976-989.2006

    Figure Lengend Snippet: Characterization of part of the mouse Rad54B genomic locus and generation of mouse ES cells carrying a disrupted mRad54B allele. (A) Part of the mRad54B genomic locus and structure of the targeting construct. Exons 12 to 15 are indicated by black boxes. Shown are the locations of selected restriction sites, EcoRI (E), BamHI (B), BglII (Bg), HindIII (H), and XbaI (X). The positions of two different probes, named A and B, are indicated. (B) DNA blot analysis of G418-resistant ES clones with probe A and EcoRI digested DNA. The wild-type (wt) allele yields a 3.0-kb band, while the disrupted allele results in a 3.6-kb band. Lane 1, wild-type ES cell; lane 2, clone with a randomly integrated targeting construct; lane 3, clone with a homologously integrated targeting construct. (C) RNA blot analysis of mRad54B transcripts in mice carrying the disrupted allele. Total RNA (15 μg) isolated from testes of wild-type, mRad54B +/ − , and mRad54B −/− males was probed with 5′ and 3′ mRad54B cDNA probes. A GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA probe served as a loading control.

    Article Snippet: The hRAD54B cDNA was subcloned from pUC18 into the BamHI site of pFastBac (Invitrogen).

    Techniques: Construct, Clone Assay, Northern blot, Mouse Assay, Isolation

    Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Journal: Jundishapur Journal of Microbiology

    Article Title: Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance

    doi: 10.5812/jjm.29384

    Figure Lengend Snippet: Confirmation of Self-Ligated pP15A, kana R by Digestion Lane 2: digestion of pP15A, kana R vector with BamHI ; lane 3, xbaI; Lane 4, double digestion with BamHI-XbaI; Lane 5, undigested pP15A, kana R vector; Lanes 1 and 6, GeneRuler DNA ladder mix (Thermo Scientific, Waltham, MA, USA).

    Article Snippet: The pTZ57R plasmid was extracted using a GeneJET™ Plasmid Miniprep Kit (Thermo Scientific, Waltham, MA, USA) and was digested using EcoRI and BamHI restriction enzymes (Thermo Scientific, Waltham, MA, USA).

    Techniques: Plasmid Preparation

    Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Mutational specificity and genetic control of replicative bypass of an abasic site in yeast

    doi: 10.1073/pnas.0711227105

    Figure Lengend Snippet: Plasmids used for TLS assays. ( A ) Construction of an AP site-containing plasmid. Plasmid pVP15 is linearized by BbrPI digestion. The PstI-BamHI fragment of pVP9 is removed by digestion and purification on a microcon YM-30 column. Both linearized plasmids

    Article Snippet: Monoadducted heteroduplex plasmids were generated as follows: Plasmids pVP9 and pVP10 were digested with BamHI and PstI ( A ), and the vector portion was separated from the 25-bp insert by centrifugation in a YM-30 microcon filter unit (Millipore).

    Techniques: Plasmid Preparation, Purification

    Integron comparisons based on BamHI digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696

    Journal: Antimicrobial Resistance and Infection Control

    Article Title: Characterization of the novel In1059 harbouring VIM gene cassette

    doi: 10.1186/s13756-017-0204-1

    Figure Lengend Snippet: Integron comparisons based on BamHI digestion of plasmids. Genes are denoted by arrows and colours based on the gene function classifications. Shaded areas denote regions with > 99% nucleotide sequence identity, with the exception of > 97% for the comparison of GC aacA4cr in In846 and GC aacA4′-37 in novel In1059. A , Comparison of In37, In843, In62, In1021, In846 and novel In1059 integrons after BamHI digestion. B - a , Comparison of integrons In37, novel In1059 and transposon Tn1696 (DQ310703); B - b , Comparison of integrons In846, novel In1059 and transposon Tn7017 (KJ571202). The novel In1059 integron shares higher sequence identity with integron Tn7017 than with integron Tn1696

    Article Snippet: The plasmids were digested with BamHI (TaKaRa, Dalian, China), and the six integron-harbouring recombinant plasmids were electrophoretically resolved to generate genetic maps.

    Techniques: Sequencing

    Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Journal: The Journal of Clinical Investigation

    Article Title: Human herpesvirus–encoded kinase induces B cell lymphomas in vivo

    doi: 10.1172/JCI97053

    Figure Lengend Snippet: Generation of vPK transgenic mice. FLAG-tagged vPK was cloned, using HinDIII-HF and BamHI-HF, into a plasmid containing a Ub- and hGH-stabilization element. The linearized transgene fragment was microinjected into the embryos of C57BL/6 mice and implanted into pseudopregnant female mice. Each vPK line was developed from breeding a vPK founder to a C57BL/6 mouse. ( A ) Schematic of the linearized construct used to generate the vPK transgenic mice, as well as the cut sites and probe for the Southern blot in D . ( B ) DNA from the tails of 2 WT (wA and wB) and 2 vPK transgenic mice (vA and vB) for lines 1 and 2 was isolated and evaluated by PCR for the vPK transgene. ( C ) Expression of vPK protein in lysates from spleens of the mice in B as determined by SDS-PAGE and Western blot. Lysate from HEK-293 cells that transiently express vPK was used as a positive control for vPK expression. ( D ) Southern blot of WT, vPK1, and vPK2. DNA was isolated from the spleens of WT and vPK lines 1 and 2. D, double digest with HinDIII-HF and BamHI-HF; S, single digestion with AFlII; p-vPK, plasmid Ub.vPK.hGH.

    Article Snippet: Fifteen micrograms of DNA from each mouse was double digested with BamHI-HF and HindIII-HF (New England BioLabs) and single digested using AflII (New England BioLabs) in a total volume of 100 μl at 37°C overnight.

    Techniques: Transgenic Assay, Mouse Assay, Clone Assay, Plasmid Preparation, Construct, Southern Blot, Western Blot, Isolation, Polymerase Chain Reaction, Expressing, SDS Page, Positive Control

    Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: Umbravirus 3′ UTRs contain a BTE. (A) Representative predicted (Mfold) secondary structure of the BTE in the 3′ UTR of TBTV genomic RNA, and relative translation activities of selected mutants. 17 nt CS is in bold italics. The shaded bases were deleted in mutants dm1 and dm2 as indicated. In mutants sm1 and sm2, bases in dashed boxes were replaced by bases in solid boxes (dashed arrows). The luciferase activity generated by translation of uncapped mutant RNAs, as a percentage of wild type (100%) are indicated. (B) Genome organization of TBTV RNA and maps of translation reporter constructs containing TBTV UTRs. TUlucTU contains both complete UTRs of TBTV flanking the firefly luciferase coding sequence (fLUC). TUluc has only the TBTV 5′UTR. TUlucTUBF differs from TUlucTU by only a 4 nt insertion (underlined) in the BamH I site. Bases are numbered according to their position in the TBTV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities obtained from the indicated RNAs are normalized to uncapped TUlucTU (defined as 100%) and shown as RLU (relative luciferase units). Error bars indicate standard error. (D) Predicted secondary structure of the GRV BTE. The 17 nt CS (italics) is in the dashed box, with bases that deviate from consensus indicated in bold italics. Mutant sequence (CS) which is identical to the consensus 17 nt CS is indicated at right by dashed arrow. (E) Map of reporter RNA GlucG. GlucG contains the GRV 5′UTR and a chimeric 3′UTR consisting of the TBTV 3′ UTR with the putative GRV BTE in place of the TBTV BTE. Positions of bases from each virus are indicated. (F) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Luciferase, Activity Assay, Generated, Mutagenesis, Construct, Sequencing

    ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Journal: Virology

    Article Title: Structural plasticity of Barley yellow dwarf virus-like cap-independent translation elements in four genera of plant viral RNAs

    doi: 10.1016/j.virol.2010.03.025

    Figure Lengend Snippet: ). 17 nt CS is shown in bold italic. (B) Genome organization of RSDaV RNA and maps of translation reporter constructs. RSlucRS has both UTRs of RSDaV flanking the luciferase coding sequence (fLUC). RSluc has only the RSDaV 5′UTR. RSlucRSBF differs from RSlucRS only by a 4 nt GAUC insertion (underlined) in the BamH I site. Bases are numbered according to their position in the RSDaV genome. (C) Relative translation activities of capped and uncapped reporter mRNAs in wheat germ extract. Luciferase activities are normalized to uncapped RSlucRS (defined as 100%). Error bars indicate standard error.

    Article Snippet: It was generated by digesting with BamH I and then filling in the sticky ends with DNA polymerase Pfx (Invitrogen) followed by blunt-end re-ligation. pGlucG was derived by inserting the PCR-amplified 12 nt 5′UTR of GRV into EcoR I/Sca I-digested pTUlucTU, in place of the TBTV 5′ UTR.

    Techniques: Construct, Luciferase, Sequencing

    Radioactive RT-PCR assay for detecting the use of alternative 3′ splice sites. The assay was conducted with exon-specific primers I4B_right (radiolabeled) and 5p_left_BamHI ( Table 1 ) with wild-type (WT) and d3ps mutant constructs spliced in vitro in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 and 100 mM MgCl 2 at 45°C. While no alternative splicing was detected for the WT B.c .I4 intron, the d3ps mutant could use a 3′ splice site at position +2 downstream of the mutated wild-type site. Quantification of the bands using a phosphorimager is shown, expressed as percentage of total radioactivity. The intron-3′ exon splice junction is shown on the right, with mutated nucleotides boxed.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: Radioactive RT-PCR assay for detecting the use of alternative 3′ splice sites. The assay was conducted with exon-specific primers I4B_right (radiolabeled) and 5p_left_BamHI ( Table 1 ) with wild-type (WT) and d3ps mutant constructs spliced in vitro in 40 mM MOPS (pH 7.5), 500 mM (NH 4 ) 2 SO 4 and 100 mM MgCl 2 at 45°C. While no alternative splicing was detected for the WT B.c .I4 intron, the d3ps mutant could use a 3′ splice site at position +2 downstream of the mutated wild-type site. Quantification of the bands using a phosphorimager is shown, expressed as percentage of total radioactivity. The intron-3′ exon splice junction is shown on the right, with mutated nucleotides boxed.

    Article Snippet: Plasmid constructs for in vitro self-splicing experiments were made by cloning a PCR product covering the entire B.c .I4 intron and parts of the flanking exons, amplified with primers 5p_left_BamHI and 3p_right_ClaI , into the BamHI site of pBluescript II KS+ (Stratagene), in orientation for T7 transcription.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Construct, In Vitro, Radioactivity

    Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.

    Journal: Microbial biotechnology

    Article Title: T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2008.00029.x

    Figure Lengend Snippet: Southern blot analysis of L. bicolor transgenic and wild‐type strains. Total DNA (7 µg) was digested with BamHI which cuts once within the T‐DNA, blotted and probed with the ∼1 kb amp gene fragment. From left to right: molecular size marker λ BstE II, L. bicolor wild type (Wt) and transgenic strains.

    Article Snippet: Southern blotting Approximately 7 µg of fungal genomic DNA per sample was digested overnight with BamHI (Promega) which cuts once within the T‐DNA, separated in 1% agarose gel and transferred by alkaline capillarity blotting to a Hygrobond‐ N+ nylon membrane (Amershamn Biosciences) according to the manufacturer's protocol.

    Techniques: Southern Blot, Transgenic Assay, Marker

    pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus : glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus . hph : hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.

    Journal: Microbial biotechnology

    Article Title: T-DNA insertion, plasmid rescue and integration analysis in the model mycorrhizal fungus Laccaria bicolor

    doi: 10.1111/j.1751-7915.2008.00029.x

    Figure Lengend Snippet: pHg/pBks rescue plasmid. The binding site for the Post‐RB primer is indicated by an arrow. GPD promoter Agaricus bisporus : glyceraldehide‐3‐phosphate dehydrogenase promoter of Agaricus bisporus . hph : hph gene of E. coli coding for an aminocyclitol phosphotransferase that confers resistance to Hygromycin B and structurally related antibiotics. 35S‐3′: cauliflower mosaic virus 35S terminator. Amp R: bla (ApR) gene of E. coli coding for a β‐lactamase that confers resistance to ampicillin. ORI: replication origin of pBluescript KS+. LB: T‐DNA left border of pCAMBIA1300. RB: T‐DNA right border of pCAMBIA1300. Kan R: aadA gene of E. coli coding for an aminoglycoside phosphotransferase that confers resistance to kanamycin. Relevant restriction sites (SacI, BamHI and XbaI) within the T‐DNA are indicated.

    Article Snippet: Southern blotting Approximately 7 µg of fungal genomic DNA per sample was digested overnight with BamHI (Promega) which cuts once within the T‐DNA, separated in 1% agarose gel and transferred by alkaline capillarity blotting to a Hygrobond‐ N+ nylon membrane (Amershamn Biosciences) according to the manufacturer's protocol.

    Techniques: Plasmid Preparation, Binding Assay