ecori hf  (New England Biolabs)


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    EcoRI HF
    Description:
    EcoRI HF 50 000 units
    Catalog Number:
    r3101l
    Price:
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    Size:
    50 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs ecori hf
    EcoRI HF
    EcoRI HF 50 000 units
    https://www.bioz.com/result/ecori hf/product/New England Biolabs
    Average 99 stars, based on 3355 article reviews
    Price from $9.99 to $1999.99
    ecori hf - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    2) Product Images from "Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae"

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133654

    Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.
    Figure Legend Snippet: Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Techniques Used: Plasmid Preparation

    3) Product Images from "aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis"

    Article Title: aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis

    Journal: Nature Communications

    doi: 10.1038/ncomms11643

    Lgl regulates the length of microridges by promoting their fusion. Live time-lapse imaging of microridge elongation ( a ) in clones expressing lifeActRFP under CMV promoter in wild-type and lgl1 morphant embryos during 23–30 hpf. Immunolocalization of Lgl and F-actin at the basolateral domain in wild-type and pen/lgl2 mutant at 48 hpf ( b ). Confocal images of immunostaining using anti-GFP antibody and phalloidin in wild-type embryos-injected with eGFP-xLgl2 construct under CMV promoter at 24 hpf ( c ) and their orthogonal sections ( d ). Visualization of the distribution of the ridge lengths and medians in eGFP-xLgl2 expressing clones and surrounding non-GFP cells using bean plots ( e ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories ( f ). The box-whisker plots ( g ) represent the distributions of means and variances per cell in eGFP-xLgl2 expressing clones and surrounding non-GFP cells. Data presented in e – g is based on ridge-length measurements done on phalloidin stainings performed in the eGFP-xLgl2 injected embryos at 24 hpf. In bean plots ( e ) and box-whisker plots ( g ) the alphabets ‘a' and ‘b' represent significant difference in median values at P
    Figure Legend Snippet: Lgl regulates the length of microridges by promoting their fusion. Live time-lapse imaging of microridge elongation ( a ) in clones expressing lifeActRFP under CMV promoter in wild-type and lgl1 morphant embryos during 23–30 hpf. Immunolocalization of Lgl and F-actin at the basolateral domain in wild-type and pen/lgl2 mutant at 48 hpf ( b ). Confocal images of immunostaining using anti-GFP antibody and phalloidin in wild-type embryos-injected with eGFP-xLgl2 construct under CMV promoter at 24 hpf ( c ) and their orthogonal sections ( d ). Visualization of the distribution of the ridge lengths and medians in eGFP-xLgl2 expressing clones and surrounding non-GFP cells using bean plots ( e ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories ( f ). The box-whisker plots ( g ) represent the distributions of means and variances per cell in eGFP-xLgl2 expressing clones and surrounding non-GFP cells. Data presented in e – g is based on ridge-length measurements done on phalloidin stainings performed in the eGFP-xLgl2 injected embryos at 24 hpf. In bean plots ( e ) and box-whisker plots ( g ) the alphabets ‘a' and ‘b' represent significant difference in median values at P

    Techniques Used: Imaging, Clone Assay, Expressing, Mutagenesis, Immunostaining, Injection, Construct, Whisker Assay

    Apically localized Lgl promotes an increase in the microridge length. Confocal sections at the apical ( z =2) and basolateral level ( z =7) of the peridermal cells stained ( a – c ) for GFP and F-actin at 30 hpf in wild-type embryos injected with eGFP-mLgl1 ( a ) eGFP-Ezrin ( b ) and eGFP-Ezrin-mLgl1 ( c ) under CMV promoter along with their corresponding orthogonal sections. Visualization of the distribution of ridge lengths and medians–estimated from clones expressing eGFP-mLgl1 ( d ) eGFP-Ezrin ( e ) and eGFP-Ezrin-mLgl1 ( f ) and their corresponding non-GFP controls using bean plots. Comparison between the ridge lengths exhibited by clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories for clones expressing eGFP-mLgl1 ( d′ ), eGFP-Ezrin ( e′ ) and eGFP-Ezrin-mLgl1 ( f′ ) along with their corresponding non-GFP controls. The comparison between frequency distributions observed in clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g′ ). Quantifications in ( d – g ) and ( d′ – g′ ) are based on phalloidin stainings performed at 30 hpf in the embryos injected with the above mentioned eGFP constructs. Note the minimal localization of eGFP-Ezrin-mLgl1 and eGFP Ezrin to the basolateral cortex as compared with eGFP-Lgl1. The distributions represented by two different alphabets in d – g show significant difference at P
    Figure Legend Snippet: Apically localized Lgl promotes an increase in the microridge length. Confocal sections at the apical ( z =2) and basolateral level ( z =7) of the peridermal cells stained ( a – c ) for GFP and F-actin at 30 hpf in wild-type embryos injected with eGFP-mLgl1 ( a ) eGFP-Ezrin ( b ) and eGFP-Ezrin-mLgl1 ( c ) under CMV promoter along with their corresponding orthogonal sections. Visualization of the distribution of ridge lengths and medians–estimated from clones expressing eGFP-mLgl1 ( d ) eGFP-Ezrin ( e ) and eGFP-Ezrin-mLgl1 ( f ) and their corresponding non-GFP controls using bean plots. Comparison between the ridge lengths exhibited by clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories for clones expressing eGFP-mLgl1 ( d′ ), eGFP-Ezrin ( e′ ) and eGFP-Ezrin-mLgl1 ( f′ ) along with their corresponding non-GFP controls. The comparison between frequency distributions observed in clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g′ ). Quantifications in ( d – g ) and ( d′ – g′ ) are based on phalloidin stainings performed at 30 hpf in the embryos injected with the above mentioned eGFP constructs. Note the minimal localization of eGFP-Ezrin-mLgl1 and eGFP Ezrin to the basolateral cortex as compared with eGFP-Lgl1. The distributions represented by two different alphabets in d – g show significant difference at P

    Techniques Used: Staining, Injection, Clone Assay, Expressing, Construct

    4) Product Images from "aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis"

    Article Title: aPKC regulates apical localization of Lgl to restrict elongation of microridges in developing zebrafish epidermis

    Journal: Nature Communications

    doi: 10.1038/ncomms11643

    Apically localized Lgl promotes an increase in the microridge length. Confocal sections at the apical ( z =2) and basolateral level ( z =7) of the peridermal cells stained ( a – c ) for GFP and F-actin at 30 hpf in wild-type embryos injected with eGFP-mLgl1 ( a ) eGFP-Ezrin ( b ) and eGFP-Ezrin-mLgl1 ( c ) under CMV promoter along with their corresponding orthogonal sections. Visualization of the distribution of ridge lengths and medians–estimated from clones expressing eGFP-mLgl1 ( d ) eGFP-Ezrin ( e ) and eGFP-Ezrin-mLgl1 ( f ) and their corresponding non-GFP controls using bean plots. Comparison between the ridge lengths exhibited by clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories for clones expressing eGFP-mLgl1 ( d′ ), eGFP-Ezrin ( e′ ) and eGFP-Ezrin-mLgl1 ( f′ ) along with their corresponding non-GFP controls. The comparison between frequency distributions observed in clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g′ ). Quantifications in ( d – g ) and ( d′ – g′ ) are based on phalloidin stainings performed at 30 hpf in the embryos injected with the above mentioned eGFP constructs. Note the minimal localization of eGFP-Ezrin-mLgl1 and eGFP Ezrin to the basolateral cortex as compared with eGFP-Lgl1. The distributions represented by two different alphabets in d – g show significant difference at P
    Figure Legend Snippet: Apically localized Lgl promotes an increase in the microridge length. Confocal sections at the apical ( z =2) and basolateral level ( z =7) of the peridermal cells stained ( a – c ) for GFP and F-actin at 30 hpf in wild-type embryos injected with eGFP-mLgl1 ( a ) eGFP-Ezrin ( b ) and eGFP-Ezrin-mLgl1 ( c ) under CMV promoter along with their corresponding orthogonal sections. Visualization of the distribution of ridge lengths and medians–estimated from clones expressing eGFP-mLgl1 ( d ) eGFP-Ezrin ( e ) and eGFP-Ezrin-mLgl1 ( f ) and their corresponding non-GFP controls using bean plots. Comparison between the ridge lengths exhibited by clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g ). The frequency distribution of ridges in short (0–5 μm), intermediate (5–20 μm), long (20–100 μm) and very long ( > 100 μm) categories for clones expressing eGFP-mLgl1 ( d′ ), eGFP-Ezrin ( e′ ) and eGFP-Ezrin-mLgl1 ( f′ ) along with their corresponding non-GFP controls. The comparison between frequency distributions observed in clones expressing eGFP-Ezrin, eGFP-mLgl1 and eGFP-Ezrin-mLgl1 ( g′ ). Quantifications in ( d – g ) and ( d′ – g′ ) are based on phalloidin stainings performed at 30 hpf in the embryos injected with the above mentioned eGFP constructs. Note the minimal localization of eGFP-Ezrin-mLgl1 and eGFP Ezrin to the basolateral cortex as compared with eGFP-Lgl1. The distributions represented by two different alphabets in d – g show significant difference at P

    Techniques Used: Staining, Injection, Clone Assay, Expressing, Construct

    5) Product Images from "Efficient Dual-Negative Selection for Bacterial Genome Editing"

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing

    Journal: bioRxiv

    doi: 10.1101/2020.03.03.974816

    An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.
    Figure Legend Snippet: An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.

    Techniques Used: Plasmid Preparation, Clone Assay, Selection, Mutagenesis, Polymerase Chain Reaction

    6) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    7) Product Images from "Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans"

    Article Title: Multiple Phenotypes Resulting from a Mutagenesis Screen for Pharynx Muscle Mutations in Caenorhabditis elegans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026594

    PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.
    Figure Legend Snippet: PAS136 mapping and pharynx markers. (A) Probable location of the PAS136 pharynx phenotype allele is between 6 cM and 8 cM on LG.I relative to the genetic center of the chromosome (green circle) derived by mapping with DraI or EcoRI specific SNPs corresponding to DNA clones D1007, K02B12, B0205, and F58D5 (orange lines) and between 4.64 cM and 9.2 cM (red circle) using complementation with the deficiency strains MT2179, DC1079, KR2838 and SL536 with overlapping chromosomal deletions (blue lines). (B) pha-4 RNAi used a positive control for pharynx phenotypes, arrow shows lack of myo-2::GFP in most of the head. (C) lam-3 (T22A3.8) RNAi showing a phenotype similar to PAS136 with non-adherent cells (arrow). (D) blmp-1 (F25D7.3) RNAi has a less severe PAS136 phenotype (arrow denotes cell disconnected from the pharynx). (E) hmr-1 (W02B9.1) RNAi results in a Pun phenotype with diminished anterior pharynx cells (arrow). (F) Wild-type MH27 AJM-1 adherens junction antibody staining showing pharynx (ph) and intestine (it) localization. (G) PAS136 embryo with weak and disconnect AJM-1 staining in the pharynx (ph) and more normal AJM-1 in the intestine (it). (H) Wild-type Intermediate Filaments showing three sets of marginal cells (arrows). (I) PAS136 embryo with three sets of marginal cells (arrows). Bar is ∼10 µM.

    Techniques Used: Derivative Assay, Clone Assay, Positive Control, Laser Capture Microdissection, Staining

    8) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
    Figure Legend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    9) Product Images from "Transcription-induced formation of extrachromosomal DNA during yeast ageing"

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000471

    Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.
    Figure Legend Snippet: Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.

    Techniques Used: Southern Blot, Derivative Assay, Plasmid Preparation, Transformation Assay, Colony Assay, DNA Sequencing

    10) Product Images from "Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology"

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology

    Journal: eLife

    doi: 10.7554/eLife.52741

    Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.
    Figure Legend Snippet: Mapping T-DNA genomic insertion sites with inverse PCR. ( A ) Diagram of T-DNA integration and the location of PCR/sequencing primers and restriction sites used for inverse PCR (invPCR). We only show primers adjacent to the left border (LB) because they consistently amplified for all transformants, unlike the primers adjacent to the right border. ( B ) Example of amplification by invPCR of the LB-genome border after EcoRI genomic digestion and ligation for an untransformed strain (WT), four independent transformants and non-template control. ( C ) Amplification by invPCR of the LB-genome border after HindIII genomic digestion and ligation. T-DNA location for all transformants was confirmed by two independent biological replicates (i.e. independent genomic extractions, ligation and invPCR). ( D ) T-DNA insertion sites in four independent transformants of Spizellomyces . In strain EM20C-3, invPCR for EcoRI indicated LB is located toward SPPG_02523, while invPCR for HindIII shows same insertion site but with an inverted direction. The divergent invPCR results might represent an insertion of a tandem inverted T-DNA. ( E ) Three of the four strains (EM20C-2,3,4) have similar tdTomato fluorescence levels as determined by flow cytometry.

    Techniques Used: Inverse PCR, Polymerase Chain Reaction, Sequencing, Amplification, Ligation, Fluorescence, Flow Cytometry

    11) Product Images from "Efficient dual-negative selection for bacterial genome editing"

    Article Title: Efficient dual-negative selection for bacterial genome editing

    Journal: BMC Microbiology

    doi: 10.1186/s12866-020-01819-2

    An optimized method for genome editing in Salmonella enterica . a Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter; R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci ( sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [ 30 ]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection
    Figure Legend Snippet: An optimized method for genome editing in Salmonella enterica . a Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter; R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci ( sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [ 30 ]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection

    Techniques Used: Plasmid Preparation, Clone Assay, Selection, Mutagenesis, Polymerase Chain Reaction

    12) Product Images from "Depurination of colibactin-derived interstrand cross-links"

    Article Title: Depurination of colibactin-derived interstrand cross-links

    Journal: bioRxiv

    doi: 10.1101/869313

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).
    Figure Legend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Techniques Used: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis

    13) Product Images from "Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme"

    Article Title: Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0076-6

    L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene
    Figure Legend Snippet: L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene

    Techniques Used: Functional Assay, Construct, Mutagenesis, Activity Assay, Cell Culture, Staining, Expressing

    14) Product Images from "Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions"

    Article Title: Long-Distance Phasing of a Tentative “Enhancer” Single-Nucleotide Polymorphism With CYP2D6 Star Allele Definitions

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2020.00486

    Mile Post experiment to establish single-nucleotide polymorphism (SNP) linkage. SNPs at increased distances relative to the CYP2D6*2 core SNP (rs16947) were interrogated to establish DropPhase2D6. The percent (%) linkage between SNPs is decreasing as the distance between interrogated SNPs increases. SNPs that are trans -configured show no/little linkage (e.g., rs16947A and rs5758550G). No linkage was observed when DNA was pre-treated with the restriction enzyme Eco RI. n/c, negative control, i.e., genotype does not support signal generation with respective probe/assay combinations.
    Figure Legend Snippet: Mile Post experiment to establish single-nucleotide polymorphism (SNP) linkage. SNPs at increased distances relative to the CYP2D6*2 core SNP (rs16947) were interrogated to establish DropPhase2D6. The percent (%) linkage between SNPs is decreasing as the distance between interrogated SNPs increases. SNPs that are trans -configured show no/little linkage (e.g., rs16947A and rs5758550G). No linkage was observed when DNA was pre-treated with the restriction enzyme Eco RI. n/c, negative control, i.e., genotype does not support signal generation with respective probe/assay combinations.

    Techniques Used: Negative Control

    15) Product Images from "Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern"

    Article Title: Multiple Pairwise Analysis of Non-homologous Centromere Coupling Reveals Preferential Chromosome Size-Dependent Interactions and a Role for Bouquet Formation in Establishing the Interaction Pattern

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006347

    3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.
    Figure Legend Snippet: 3C2D-qPCR design for characterizing centromere coupling. (A) Design of two primers (arrow) and one Taqman probe (ball-and-stick) to quantify the interaction between restriction fragments ligated together, each encompassing a non-homologous centromere (oval). (B) Distribution of restriction enzyme sites on fragments encompassing the centromere ( CEN ) on all 16 chromosomes using an EcoRI single digestion (3C) (left) or an EcoRI-MfeI double digestion (3C2D) (right). For each chromosome (on y-axis), the distances of the restriction sites delimitating the CEN fragment are given in kilobases (kb), in relation to the center of the CEN (x-axis). Blue vertical lines indicate EcoRI sites and red lines indicate MfeI sites.

    Techniques Used: Real-time Polymerase Chain Reaction

    16) Product Images from "RNA-DNA hybrids support recombination-based telomere maintenance in fission yeast"

    Article Title: RNA-DNA hybrids support recombination-based telomere maintenance in fission yeast

    Journal: bioRxiv

    doi: 10.1101/458968

    Degradation of RNA-DNA hybrids induces a second growth crisis in ccq1Δ survivors, ultimately leading to a new type of RNase H-resistant ccq1Δ survivor. (A) Rnh201 induction returns ccq1Δ survivors into a growth crisis. WT or ccq1Δ Taz1-GFP survivor cells (MKSP1213 and MKSP1214) were modified to insert the nmt41 promoter upstream of the Rnh201 coding sequence. Strains were then cultured in a repressed state (with thiamine) or induced state (without thiamine), as indicated and diluted every 24 hours. (B) DIC imaging shows that Rnh201 over-expression leads to a new growth crisis in ccq1Δ survivors. The first three days of liquid culturing corresponding to Figure 3A are shown. Note the increase in cell length, indicating checkpoint arrest. (C,D) The telomeres of RHR ccq1Δ survivors selected after constitutive Rnh201 over-expression are similar in length and structure to the initial ccq1Δ survivors. Southern blot of EcoRI digested genomic DNA as in Fig. S1B. The rDNA probe serves as a loading control. (D) The RHR ccq1Δ survivors, like ccq1Δ survivors, retain linear chromosomes as assessed by pulsed-field gel electrophoresis followed by Southern blotting. Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes, which detect the terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated by M+C and I+L bands, observed in the trt1Δ and ccq1Δrad55Δ circular survivors.
    Figure Legend Snippet: Degradation of RNA-DNA hybrids induces a second growth crisis in ccq1Δ survivors, ultimately leading to a new type of RNase H-resistant ccq1Δ survivor. (A) Rnh201 induction returns ccq1Δ survivors into a growth crisis. WT or ccq1Δ Taz1-GFP survivor cells (MKSP1213 and MKSP1214) were modified to insert the nmt41 promoter upstream of the Rnh201 coding sequence. Strains were then cultured in a repressed state (with thiamine) or induced state (without thiamine), as indicated and diluted every 24 hours. (B) DIC imaging shows that Rnh201 over-expression leads to a new growth crisis in ccq1Δ survivors. The first three days of liquid culturing corresponding to Figure 3A are shown. Note the increase in cell length, indicating checkpoint arrest. (C,D) The telomeres of RHR ccq1Δ survivors selected after constitutive Rnh201 over-expression are similar in length and structure to the initial ccq1Δ survivors. Southern blot of EcoRI digested genomic DNA as in Fig. S1B. The rDNA probe serves as a loading control. (D) The RHR ccq1Δ survivors, like ccq1Δ survivors, retain linear chromosomes as assessed by pulsed-field gel electrophoresis followed by Southern blotting. Genomic DNA was digested with NotI and hybridized to C, I, L, and M probes, which detect the terminal fragments of chromosomes I and II. Bands corresponding to chromosome end fusions are indicated by M+C and I+L bands, observed in the trt1Δ and ccq1Δrad55Δ circular survivors.

    Techniques Used: Modification, Sequencing, Cell Culture, Imaging, Over Expression, Southern Blot, Pulsed-Field Gel, Electrophoresis

    17) Product Images from "Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology"

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    Journal: Nature protocols

    doi: 10.1038/nprot.2018.012

    Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.
    Figure Legend Snippet: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Techniques Used: CRISPR, Non-Homologous End Joining, Mutagenesis, Mouse Assay, Generated, Electroporation

    18) Product Images from "Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme"

    Article Title: Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0076-6

    L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene
    Figure Legend Snippet: L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene

    Techniques Used: Functional Assay, Construct, Mutagenesis, Activity Assay, Cell Culture, Staining, Expressing

    19) Product Images from "An exogenous chloroplast genome for complex sequence manipulation in algae"

    Article Title: An exogenous chloroplast genome for complex sequence manipulation in algae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1008

    Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.
    Figure Legend Snippet: Characterization of the cloned C. reinhardtii chloroplast genome in vivo . ( A ) A nested set representing the presence of increasing numbers of markers in primary transformants of pCr03 into a psbD knockout strain as determined by PCR ( Table 2 ; primers used as follows: M1, 11 606 and 11 607; M2, 5512 and 5513; M3, 11 456 and 11 457; and M4, 14 067 and 14 068.). The broken circle shows the subset of transformants with M1, M2, M3 and M4 that gave rise to the same genotype upon rescreening. ( B–E ) Southern blot analysis of EcoRI (B, C and E) or NdeI (D) digests (see ‘Materials and Methods’ section). Probes were specific for sequences adjacent to integration sites for M1 (B), M2 (C), M3 (D) and M4 (E). All samples are arranged as follows: Lane L, 1 kb DNA ladder (Invitrogen; Carlsbad, CA); lane 1, wild-type; lane 2, purified pCr03; and lane 3, a representative algae clone containing all unique markers. A single band in lane 3 indicates homoplasmic integration of the marker, while two bands indicate heteroplasmy with the wild-type locus.

    Techniques Used: Clone Assay, In Vivo, Knock-Out, Polymerase Chain Reaction, Southern Blot, Purification, Marker

    20) Product Images from "Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme"

    Article Title: Evidence for L1-associated DNA rearrangements and negligible L1 retrotransposition in glioblastoma multiforme

    Journal: Mobile DNA

    doi: 10.1186/s13100-016-0076-6

    Characterisation of a somatic L1 mutation within EGFR.  a  Patient #8 EGFR mutant allele: a 0.5 kb L1-Ta sequence antisense to EGFR. Direction of transcription is indicated with  blue arrow .  b  L1 mutation magnified view: RC-seq reads detected at the L1 3' terminus ( black/red bars ). The L1 mutation comprised a truncated fragment of L1 ORF2 ( white box ) and the 3′UTR without a poly-A tail ( red box ). A 550 nucleotide deletion at the integration site was also identified (triangle). Primers used for PCR validation are indicated as pink and purple arrows.  c  Mutation site PCR validation: Region comprising the EGFR-L1 5' junction was detected in patient #8 tumour sample. No amplification was detected when water (NTC) or genomic DNA from blood were used as template.  d  qRT-PCR measurement of EGFR transcription at its 5'UTR and exon 11-to-12 junction (E 11–12): The relative levels of RNA from both regions were significantly increased in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to adjacent brain values, pooled and presented as mean +/− SEM (* p
    Figure Legend Snippet: Characterisation of a somatic L1 mutation within EGFR. a Patient #8 EGFR mutant allele: a 0.5 kb L1-Ta sequence antisense to EGFR. Direction of transcription is indicated with blue arrow . b L1 mutation magnified view: RC-seq reads detected at the L1 3' terminus ( black/red bars ). The L1 mutation comprised a truncated fragment of L1 ORF2 ( white box ) and the 3′UTR without a poly-A tail ( red box ). A 550 nucleotide deletion at the integration site was also identified (triangle). Primers used for PCR validation are indicated as pink and purple arrows. c Mutation site PCR validation: Region comprising the EGFR-L1 5' junction was detected in patient #8 tumour sample. No amplification was detected when water (NTC) or genomic DNA from blood were used as template. d qRT-PCR measurement of EGFR transcription at its 5'UTR and exon 11-to-12 junction (E 11–12): The relative levels of RNA from both regions were significantly increased in tumour ( blue ) versus adjacent brain ( green ) samples. Data for each group were normalised to adjacent brain values, pooled and presented as mean +/− SEM (* p

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR

    L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene
    Figure Legend Snippet: L1 retrotransposition rarely occurs in GBM cell lines. a Schematic representing L1 retrotransposition assay. A full-length L1 (L1.3) [ 61 ] is located upstream of the antisense oriented blasticidin resistance gene ( red boxes ). The L1 internal promoter is represented by an arrow on the 5'UTR region. Two L1 open reading frames (ORF1 and ORF2) are indicated by blue and green boxes , respectively. Functional domains of ORF2, endonuclease (EN), reverse transcriptase (RT) and cysteine rich domain (C) are also indicated. The blasticidin resistance gene is interrupted by an intron in the same orientation as the L1. Splice donor (SD) and splice acceptor (SA) sites are indicated. Polyadenylation signals are denoted by grey lollipops. b Schematic representation of retrotransposition assay constructs. JJ L1.3 WT contains an external promoter (cytomegalovirus promoter, CMV) upstream of a full length retrotransposition-competent L1.3 element [ 61 ]. Asterisk indicates missense mutation to abolish endonuclease activity (JJ L1.3 D205A), reverse-transcriptase activity (JJ L1.3 D702A) or both (JJ L1.3 D205A D702A). c Results of cell culture-based L1 retrotransposition assay. Each stained colony represents a cell where a retrotransposition event took place allowing the expression of the blasticidin resistance gene

    Techniques Used: Functional Assay, Construct, Mutagenesis, Activity Assay, Cell Culture, Staining, Expressing

    21) Product Images from "PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis"

    Article Title: PCNA-K164 ubiquitination facilitates origin licensing and mitotic DNA synthesis

    Journal: bioRxiv

    doi: 10.1101/2020.06.25.172361

    Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.
    Figure Legend Snippet: Generation of a PCNA K164R mutant cell line in RPE-1 using CRISPR/Cas9 A) Schematic of the human PCNA indicating that exon 5 was targeted by CRISPR-Cas9. The K164R mutation was knocked-in utilizing a donor plasmid. B) Schematic of screening PCR and expected PCR product sizes after EcoRI restriction enzyme digestion. C) Representative genotyping PCR. Not targeted (wildtype; 1426 bp), monoallelic knock-in (KIN) ( PCNA KR/- 1E4; 1426 bp, 1168 bp, 258 bp), and biallelic KIN ( PCNA KR/KR 1E12, 2B10; 1168bp, 258 bp). D) Karyotyping analysis from RPE-1 wildtype, PCNA KR/KR (1E12, 2B10) and PCNA KR/- (1E4) cell lines. Blue indicates expec ted RPE-1 karyotype. Red indicates chromosomal abnormalities. E) Western blot analyses of whole cell extracts from wildtype RPE-1, PCNA K164R , and RAD18 -/- cells for MCM2 with α-Tubulin as the loading control. Quantification of MCM2 levels normalized to loading control.

    Techniques Used: Mutagenesis, CRISPR, Plasmid Preparation, Polymerase Chain Reaction, Knock-In, Western Blot

    22) Product Images from "The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome"

    Article Title: The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw406

    Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.
    Figure Legend Snippet: Exit side DNA defines the extent that Chd1 slides nucleosomes in the presence of Lac repressor. ( A ) Nucleosomes are dynamically shifted back-and-forth by Chd1 in the presence of LacI. End-positioned 0N70 nucleosomes containing a LacO(−11) site on the 70 bp side and an EcoRI cut site just inside the 0 bp side were digested by EcoRI and monitored by SDS-PAGE. The slower rate of digestion in the absence of ATP (gray) demonstrates that the EcoRI site is initially buried. In the presence of Chd1 and ATP, the same fraction of nucleosomal DNA becomes cleaved in the presence (filled circle) or absence (open circle) of LacI, demonstrating dynamic repositioning by Chd1 in the presence of LacI. Based on single exponential fits to the data, the sliding rate in the presence of LacI was calculated to be 7-fold slower than in the absence of LacI. Error bars indicate the standard deviations from five or more independent experiments. ( B ) Comparison of nucleosome sliding reactions carried out in the absence and presence of LacI, using (−10)N80[LacO-11R] substrates. Time points for these experiments were 0, 1, 4, 16, 64 min. ( C ) Comparison of the preferred distributions of nucleosome positions for 0N80 and (−10)N80 nucleosomes when Chd1 sliding was carried out in the presence of LacI. White peaks show zero time points and gray peaks are the nucleosome positions at 64 min time points.

    Techniques Used: SDS Page

    23) Product Images from "Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora"

    Article Title: Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00341-17

    Schematic representation of the construct generated by overlap PCR for cloning into pBluescript and subsequent homologous recombination with Y2 genomic DNA. Annealing regions of the primers are depicted by small black arrows, and the restriction sites of EcoRI and BamHI, as well as the RBS (thick vertical line), are indicated. The image is not drawn to scale.
    Figure Legend Snippet: Schematic representation of the construct generated by overlap PCR for cloning into pBluescript and subsequent homologous recombination with Y2 genomic DNA. Annealing regions of the primers are depicted by small black arrows, and the restriction sites of EcoRI and BamHI, as well as the RBS (thick vertical line), are indicated. The image is not drawn to scale.

    Techniques Used: Construct, Generated, Polymerase Chain Reaction, Clone Assay, Homologous Recombination

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Article Title: Efficient dual-negative selection for bacterial genome editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1 h at 37 °C, or PCR-amplified, and purified on agarose gel. ..

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1h at 37°C, or PCR-amplified, and purified on agarose gel. ..

    Transformation Assay:

    Article Title: Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology
    Article Snippet: .. 2.5 µg of genomic DNA of wild type (WT) and the four transformed strains of Spizellomyces (EM20C-1,2,3,4) was digested in a final volume of 50μL with 100U of EcoRI-HF (NEB R3101S) or HindIII-HF (NEB R3104S) for 24 hr at 37°C. .. After assessing the quality of the digestion by gel electrophoresis, the reaction was heat inactivated and 48µL of the digested DNA was incubated with 1µL of T4 ligase (400u/µL) for 48 hr at 4°C.

    Agarose Gel Electrophoresis:

    Article Title: Efficient dual-negative selection for bacterial genome editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1 h at 37 °C, or PCR-amplified, and purified on agarose gel. ..

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1h at 37°C, or PCR-amplified, and purified on agarose gel. ..

    Plasmid Preparation:

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Purification:

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly
    Article Snippet: .. Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel). .. Ligation was mediated by T7 DNA ligase (NEB) to construct mUAV, Level 1 and Level 2 Acceptor Vectors.

    Article Title: Depurination of colibactin-derived interstrand cross-links
    Article Snippet: .. To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop. .. For each reaction with E.coli , 800 ng of linearized pUC19 DNA was added to 200 µL (6.2 µM base pairs) of M9-CA medium inoculated with 1.2 × 107 bacteria pre-grown to exponential phase in the M9-CA medium.

    Article Title: Efficient dual-negative selection for bacterial genome editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1 h at 37 °C, or PCR-amplified, and purified on agarose gel. ..

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing
    Article Snippet: .. Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1h at 37°C, or PCR-amplified, and purified on agarose gel. ..

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    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <t>PstI</t> and <t>EcoRI</t> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.
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    Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Journal: PLoS ONE

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    doi: 10.1371/journal.pone.0189892

    Figure Lengend Snippet: Proof-of-concept assembly of 16TU construct. (A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes crtE , crtB , crtI , and crtY assembled in the Vector A, the yellow chromoprotein genes scOrange , amilGFP , amajLime , and fwYellow in the Vector B, the pink chromoprotein genes tsPurple , eforRed , spisPink , and mRFP1 in the Vector Γ, and the violacein biosynthesis genes vioA , vioB , vioD and vioE in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with PstI and EcoRI resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with PstI and AleI resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.

    Article Snippet: Subsequently the pSB1C3/pSB1K3 backbones and the Golden Gate cassettes were digested with EcoRI-HF and PstI-HF (NEB) for 20 min at 37°C followed by purification with PCR Clean-up kit (Macherey Nagel).

    Techniques: Construct, Plasmid Preparation, Derivative Assay, Transformation Assay, Nucleic Acid Electrophoresis, Isolation

    Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Journal: PLoS ONE

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae

    doi: 10.1371/journal.pone.0133654

    Figure Lengend Snippet: Dendrogram of EcoRI-digested plasmids from 27 transconjugants. 15 restriction profiles were identified (P1-P15). The dashed line represents the 80% similarity level used in cluster designation. Transconjugant plasmid ID, replicon and restriction profiles are shown.

    Article Snippet: Transconjugants plasmids were digested with the restriction enzyme EcoRI -HF (New England Biolabs, UK) [ ].

    Techniques: Plasmid Preparation

    An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.

    Journal: bioRxiv

    Article Title: Efficient Dual-Negative Selection for Bacterial Genome Editing

    doi: 10.1101/2020.03.03.974816

    Figure Lengend Snippet: An optimized method for genome editing in Salmonella enterica . a ) Map of suicide plasmid pFOK ( aphA , aminoglycoside phosphotransferase gene conferring resistance to kanamycin ; I-sceI gene encoding meganuclease; oriT , origin of conjugational transfer; P tetA , tetA promoter, R6K γ ori , pi-dependent origin of replication; sacB , levansucrase gene; tetR , tetracycline repressor gene; traJ , transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing at least EcoRI, BamHI, SacI, XhoI and NotI). b-c ) Negative selection with SacB and I-SceI. b ) Mechanisms of negative selection for SacB and I-SceI, c ) Selection efficiency for various chromosomal loci ( foxA deletion, sitABCD deletion, ssrB point mutation and phoQ chimeric insertion [ 35 ]) using either SacB or I-SceI, or a combination of both. d-e ) Identification of recombination biases favoring one flanking region. d ) schematic representation of preferential recombination in the right flanking region. External primers (here primer 1 and 2) together with plasmid-specific primers (here primer oOPC-614 and oOPC-615) can be used to screen co-integrant clones to reveal such bias. e ) Recombination bias for foxA gene manipulation. PCR results of ex-conjugant screening using the primer pair 1 and oOPC-614 (left panel here oOPC-396/614) and primer 2 and oOPC-615 (right panel here oOPC-397/615). Rare ex-conjugants (here clone 5 and 10) with recombination in the non-preferred flanking region are used for subsequent counter-selection. f ) Timeframe with brief summary of daily steps.

    Article Snippet: Vectors were digested using EcoRI-HF and BamHI-HF (New England BioLabs) for 1h at 37°C, or PCR-amplified, and purified on agarose gel.

    Techniques: Plasmid Preparation, Clone Assay, Selection, Mutagenesis, Polymerase Chain Reaction

    Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Journal: bioRxiv

    Article Title: Depurination of colibactin-derived interstrand cross-links

    doi: 10.1101/869313

    Figure Lengend Snippet: Analysis of pUC19 DNA following treatment with clb − or clb + E. coli and linearization with the restriction enzyme EcoRI. The cross-linked linearized pUC19 DNA isolated from a co-culture with clb + BW25113 E. coli was used a positive control. A. Analysis of DNA by native gel electrophoresis. B. Analysis of DNA by denaturing gel electrophoresis. For both A and B: DNA ladder (Lane #1); circular pUC19 DNA standard (Lane #2); linearized pUC19 DNA standard (Lane # 3); linearized pUC19 DNA co-cultured with clb + BW25113 E. coli (Lane #4); circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli (Lane #5), reacted with buffer (Lane #6), reacted with EcoRI restriction enzyme (Lane #7); circular pUC19 DNA isolated from co-culture with clb + BW25113 E. coli (Lane #8), reacted with buffer (Lane #9), reacted with EcoRI restriction enzyme (Lane #10). Conditions (Lane #4): linearized pUC19 DNA, clb + BW25113 E. coli , M9-CA media, 4 h at 37 °C. Conditions (Lane #5–#7): circular pUC19 DNA isolated from co-culture with clb − BW25113 E. coli in M9-CA media for 4 h at 37 °C (Lane #5); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #6); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #7). Conditions (Lane #8–#10): circular pUC19 DNA isolated from co-culture with BW25113 clb + E. coli. in in M9-CA media for 4 h at 37 °C (Lane # 8); the DNA (15.4 µM base pair) was reacted with CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #9); the DNA (15.4 µM base pair) was reacted with 20 units of EcoRI-HF restriction enzyme in CutSmart Buffer® (New England Biolabs®), pH 7.9, at 37 °C for 30 minutes (Lane #10). The DNA was isolated and analyzed by native ( Fig. 5A ) or 0.4% NaOH denaturing ( Fig. 5B ) agarose gel electrophoresis (90 V, 1.5 h).

    Article Snippet: To prepare the linearized DNA, the 2686 bp pUC19 vector (New England Biolabs®) was linearized with 20 units/µg EcoRI-HF® (New England Biolabs®) and the linearized DNA was purified using a PCR clean-up kit (New England Biolabs®), eluted into 10 mM Tris (pH 8.0), and quantified using a nanodrop.

    Techniques: Isolation, Co-Culture Assay, Positive Control, Nucleic Acid Electrophoresis, Cell Culture, Agarose Gel Electrophoresis