clai  (New England Biolabs)


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    New England Biolabs clai
    Agarose gel electrophoresis analysis of restriction-enzyme-digested fEg-Eco1 9 DNA. Phage genomic DNA was digested <t>with</t> <t>EcoRI</t> (lane 2), Nsil (lane 3), SmaI (lane 4), SalI (lane 5), NruI (lane 6), <t>ClaI</t> (lane 7), AflII (lane 8), and BbvCI (lane 9). Lane 1, undigested DNA. Lane M, 1-kb DNA ladder
    Clai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86/100 stars

    Images

    1) Product Images from "Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate"

    Article Title: Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate

    Journal: Archives of Virology

    doi: 10.1007/s00705-022-05426-6

    Agarose gel electrophoresis analysis of restriction-enzyme-digested fEg-Eco1 9 DNA. Phage genomic DNA was digested with EcoRI (lane 2), Nsil (lane 3), SmaI (lane 4), SalI (lane 5), NruI (lane 6), ClaI (lane 7), AflII (lane 8), and BbvCI (lane 9). Lane 1, undigested DNA. Lane M, 1-kb DNA ladder
    Figure Legend Snippet: Agarose gel electrophoresis analysis of restriction-enzyme-digested fEg-Eco1 9 DNA. Phage genomic DNA was digested with EcoRI (lane 2), Nsil (lane 3), SmaI (lane 4), SalI (lane 5), NruI (lane 6), ClaI (lane 7), AflII (lane 8), and BbvCI (lane 9). Lane 1, undigested DNA. Lane M, 1-kb DNA ladder

    Techniques Used: Agarose Gel Electrophoresis

    2) Product Images from "Designer diatom episomes delivered by bacterial conjugation"

    Article Title: Designer diatom episomes delivered by bacterial conjugation

    Journal: Nature Communications

    doi: 10.1038/ncomms7925

    Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.
    Figure Legend Snippet: Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.

    Techniques Used: Expressing, Selection, Isolation, Agarose Gel Electrophoresis, Derivative Assay, Marker, Plasmid Preparation, Transformation Assay, Real-time Polymerase Chain Reaction, Fluorescence

    3) Product Images from "The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity"

    Article Title: The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

    Journal: bioRxiv

    doi: 10.1101/2021.11.26.470160

    eA3A activity on long, single-stranded substrates with matched sequences. (a) Left – 10 ng of ssDNA or RNA substrates were reacted with eA3A (left to right, 15 μM and then 10-fold dilutions from 6 μM to 60 pM) for 30 min at 37 °C. Following PCR or RT-PCR, the amplicons were digested with ClaI, with representative gel images shown. Right – Quantification of percent deamination as a function of A3A concentration for ssDNA (purple) or RNA (green). Data represent mean and standard deviation from four independent replicates. (b) Base resolution map showing percent deamination vs. the position of cytosines across the 720mer as per NGS analysis. ssDNA data from reaction with 0.06 nM eA3A is shown in purple above the axis, while data from RNA reacted with 6 μM eA3A is shown in green below the axis. Data represent the mean deamination at each position from two independent experiments with results from individual amplicons provided in Supplementary Table , and the most heavily deaminated cytosine for each substrate labelled with its position, selectivity factor, and percent editing.
    Figure Legend Snippet: eA3A activity on long, single-stranded substrates with matched sequences. (a) Left – 10 ng of ssDNA or RNA substrates were reacted with eA3A (left to right, 15 μM and then 10-fold dilutions from 6 μM to 60 pM) for 30 min at 37 °C. Following PCR or RT-PCR, the amplicons were digested with ClaI, with representative gel images shown. Right – Quantification of percent deamination as a function of A3A concentration for ssDNA (purple) or RNA (green). Data represent mean and standard deviation from four independent replicates. (b) Base resolution map showing percent deamination vs. the position of cytosines across the 720mer as per NGS analysis. ssDNA data from reaction with 0.06 nM eA3A is shown in purple above the axis, while data from RNA reacted with 6 μM eA3A is shown in green below the axis. Data represent the mean deamination at each position from two independent experiments with results from individual amplicons provided in Supplementary Table , and the most heavily deaminated cytosine for each substrate labelled with its position, selectivity factor, and percent editing.

    Techniques Used: Activity Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Standard Deviation, Next-Generation Sequencing

    A3A activity on long, single-stranded substrates with matched sequences. (a) 720mer assay diagram. Sequence-matched ssDNA and RNA substrates were reacted with A3A. The samples were amplified by PCR (ssDNA) or RT-PCR (RNA). Amplified products were then subjected to site-specific examination via the use of restriction enzymes, such as ClaI, or to whole amplicon Next-Generation Sequencing (NGS). ClaI cleaves non-deaminated substrates but not the deaminated products. (b) Left – Representative gels of A3A titration. 10 ng of ssDNA and RNA substrates were reacted with 10-fold dilutions of A3A (6 μM to 6 pM, left to right) for 30 min at 37 °C. Following amplification, amplicons were digested with ClaI and imaged on a 1.5% agarose gel. Right – Quantification of percent deamination at the SDHB site for ssDNA (purple) and RNA (teal) as a function of A3A concentration. Data represent four independent replicates with mean and standard deviation plotted. Product formation was fit to determine the EC 50 . (c) Base resolution map showing percent deamination vs. the position of cytosines across the 720mer as per NGS analysis. ssDNA data from reaction with 0.06 nM A3A is shown in purple above the axis, while data from RNA reacted with 60 nM A3A is shown in teal below the axis. The middle 134 bp are not included in the analysis due to the limitations of paired-end sequencing. Data represent the mean deamination at each position from two independent experiments with results from individual amplicons provided in Supplementary Table . The most heavily deaminated cytosine for each substrate is labelled with its position in the 720mer, selectivity factor, and percent editing. (d) Schematic representation of the stem-loop structures of the most heavily deaminated cytosine for each substrate. Top – cytosine at position 236 in ssDNA. Bottom – SDHB site; cytosine at position 161 in RNA. (e) Sequence logos of editing sites for ssDNA and RNA samples after correcting for background editing levels. Position 0 represents the target C. (f) Jitter plots showing percent deamination for substrates reacted with different concentrations of A3A, separated by sequence context, highlighting the higher proficiency and lower specificity for deamination of ssDNA versus RNA.
    Figure Legend Snippet: A3A activity on long, single-stranded substrates with matched sequences. (a) 720mer assay diagram. Sequence-matched ssDNA and RNA substrates were reacted with A3A. The samples were amplified by PCR (ssDNA) or RT-PCR (RNA). Amplified products were then subjected to site-specific examination via the use of restriction enzymes, such as ClaI, or to whole amplicon Next-Generation Sequencing (NGS). ClaI cleaves non-deaminated substrates but not the deaminated products. (b) Left – Representative gels of A3A titration. 10 ng of ssDNA and RNA substrates were reacted with 10-fold dilutions of A3A (6 μM to 6 pM, left to right) for 30 min at 37 °C. Following amplification, amplicons were digested with ClaI and imaged on a 1.5% agarose gel. Right – Quantification of percent deamination at the SDHB site for ssDNA (purple) and RNA (teal) as a function of A3A concentration. Data represent four independent replicates with mean and standard deviation plotted. Product formation was fit to determine the EC 50 . (c) Base resolution map showing percent deamination vs. the position of cytosines across the 720mer as per NGS analysis. ssDNA data from reaction with 0.06 nM A3A is shown in purple above the axis, while data from RNA reacted with 60 nM A3A is shown in teal below the axis. The middle 134 bp are not included in the analysis due to the limitations of paired-end sequencing. Data represent the mean deamination at each position from two independent experiments with results from individual amplicons provided in Supplementary Table . The most heavily deaminated cytosine for each substrate is labelled with its position in the 720mer, selectivity factor, and percent editing. (d) Schematic representation of the stem-loop structures of the most heavily deaminated cytosine for each substrate. Top – cytosine at position 236 in ssDNA. Bottom – SDHB site; cytosine at position 161 in RNA. (e) Sequence logos of editing sites for ssDNA and RNA samples after correcting for background editing levels. Position 0 represents the target C. (f) Jitter plots showing percent deamination for substrates reacted with different concentrations of A3A, separated by sequence context, highlighting the higher proficiency and lower specificity for deamination of ssDNA versus RNA.

    Techniques Used: Activity Assay, Sequencing, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Next-Generation Sequencing, Titration, Agarose Gel Electrophoresis, Concentration Assay, Standard Deviation

    4) Product Images from "Transduction of human embryonic stem cells by ecotropic retroviral vectors"

    Article Title: Transduction of human embryonic stem cells by ecotropic retroviral vectors

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl674

    Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.
    Figure Legend Snippet: Transduction of hES cells with murine retroviral vectors. ( A ) Transfection efficiencies of undifferentiated hES cells using optimized protocols for lipofection (L), electroporation (E) and nucleofection (N) techniques. ( B ) mCAT1-HA expression (green) in nucleofected hES cells cultured on matrigel. Twenty-four hours after plating, the cells show a flattened morphology typical for colonies propagated on matrigel. Nuclear expression of Oct-4 (red) reflects their undifferentiated state. ( C ) Schematic illustration of the two-step protocol used for transduction of hES cells with ecotropic retroviral vectors. First cells are nucleofected with a construct encoding the murine retrovirus receptor mCAT1. Twenty-four hours later they are transduced with a murine retroviral vector. Transduced cultures are either analyzed for transgene expression after 48 h or subjected to selection of permanently transduced clones. The integrated provirus expresses the EGFP transgene from the viral LTR linked to a neomycin resistance gene (neoR) by an internal ribosome entry site (IRES). ( D ) Forty-eight hours after infection, 16.4 ± 5.9% of the total cell population showed EGFP-expression. Normalized to the proportion of mCAT1-expressing cells determined in (A), this corresponds to a calculated transduction efficiency of ∼30% of the mCAT1-expressing cells (mean values from n = 5 independent experiments). ( E – G ) Transduced EGFP-positive cells continue to express the pluripotency-associated markers Oct-4 (E), Tra-1-60 (F) and Tra-1-81 (G) (all red; counterstain DAPI). ( H ) Five passages after transduction, four clones were subjected to Southern analysis. A single integration of the EGFP transgene could be detected in clones PK2, PK4 and PK7. Clone PK5 displays two bands, which could be the result of a double integration, a mixed clone population or a mutation of the vector provirus. Restriction analysis was performed with EcoRI (for genomic DNA) and ClaI (unique site within the retroviral vector). Scale bars: B,E: 100 μm; F: 30 μm; G: 50 μm.

    Techniques Used: Transduction, Transfection, Electroporation, Expressing, Cell Culture, Construct, Plasmid Preparation, Selection, Clone Assay, Infection, Mutagenesis

    5) Product Images from "Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts"

    Article Title: Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm552

    Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). ( A ) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32 P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. ( B ) Transformation efficiency of pET28a (kan R ; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (amp R ; expressed in absolute colony numbers). ( C ) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. ( D ) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.
    Figure Legend Snippet: Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). ( A ) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32 P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. ( B ) Transformation efficiency of pET28a (kan R ; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (amp R ; expressed in absolute colony numbers). ( C ) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. ( D ) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.

    Techniques Used: Incubation, Purification, Transformation Assay, Isolation

    Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). ( A ) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. ( B ) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. ( C ) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.
    Figure Legend Snippet: Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). ( A ) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. ( B ) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. ( C ) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.

    Techniques Used: Incubation, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification

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    New England Biolabs clai
    Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). <t>DNA</t> from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, <t>ClaI</t> endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.
    Clai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/clai/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    clai - by Bioz Stars, 2022-08
    95/100 stars
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    Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.

    Journal: Nature Communications

    Article Title: Designer diatom episomes delivered by bacterial conjugation

    doi: 10.1038/ncomms7925

    Figure Lengend Snippet: Demonstration that P. tricornutum episomes replicate as stable, circular, low-copy plasmids ( a – e ), and expression and localization of proteins encoded on the P. tricornutum episome p0521s (F-I). ( a ) Cultures of P. tricornutum containing p0521s (clone 9, Supplementary Fig. 3 ), were subcultured in seawater medium for 28 days with or without antibiotic selection and plated ( Supplementary Fig. 5 ). DNA from five antibiotic-resistant colonies from each culture ( Supplementary Fig. 5D ) was recovered in E. coli and isolated plasmids were separated by agarose gel electrophoresis. Shown are rescued plasmids derived from separate P. tricornutum colonies that were initially subcultured for 28 days without (lanes 1–5) or with (lanes 6–10) antibiotic selection. ‘M' designates supercoiled marker 41 and ‘C' designates the original plasmid (isolated from clone 9) introduced into P. tricornutum . Arrow denotes supercoiled plasmid band. ( b ) Stability of p0521-Se containing a 49-kb S. elongatus fragment. Ten independently transformed P. tricornutum lines containing p0521-Se were subcultured in liquid media with selection for 60 days, followed by episome rescue and separation of plasmids by agarose gel electrophoresis. Arrow denotes supercoiled plasmid band. ( c ) Plasmids extracted from P. tricornutum were untreated, treated with exonuclease, ClaI endonuclease or a combination of exonuclease and ClaI. Treated plasmids were transformed into E. coli and the number of transformed colonies was plotted (error bars indicate one s.d. of the mean from three biological replicates). ( d ) Agarose gel electrophoresis of plasmids extracted from P. tricornutum and treated with nucleases. Lanes from left to right: (1) 1 kb + ladder (NEB), (2) p0521s control (from E. coli ), (3) p0521s exonuclease-treated (extracted from P. tricornutum ), (4) p0521s untreated (extracted from P. tricornutum ), (5) 1 kb + ladder (NEB), ( e ) Copy number of p0521s in P. tricornutum determined by qPCR. Cm ( Cat gene) and His ( HIS3 gene) are loci found on the episome backbone; Ure (urease, protein ID 29702) and NR (nitrate reductase, protein ID 54983) are loci encoded on P. tricornutum nuclear chromosomes 18 and 20, respectively; Rbc (RuBisCO small subunit) and CytB (Cytochromoe B) are loci found on the P. tricornutum chloroplast and mitochondrial chromosomes, respectively. Error bars denote one s.d. of the mean from three biological replicates. ( f ) Wild-type P. tricornutum , fluorescence measured with GFP settings. ( g ) P. tricornutum expressing CFP translationally fused to beta-carbonic anhydrase (Protein ID 51305) localized to the chloroplast pyrenoid encoded on plasmid p0521s. ( h ) P. tricornutum expressing YFP translationally fused to mitochondrial urea transporter (Protein ID 39772) encoded on plasmid p0521s. ( i ) P. tricornutum expressing GFP localized to the cytoplasm encoded on plasmid p0521s. Scale bar for f – i indicates 5 μm.

    Article Snippet: First, ClaI restriction digest or mock reaction was performed on samples using 1–2 μg total DNA in a 100-μl total reaction with 1 × CutSmart buffer and ClaI (NEB) or water in digested or mock-digested samples, respectively.

    Techniques: Expressing, Selection, Isolation, Agarose Gel Electrophoresis, Derivative Assay, Marker, Plasmid Preparation, Transformation Assay, Real-time Polymerase Chain Reaction, Fluorescence

    Sequential assembly of NER factors onto RNA pol IIO. ( A ) Scheme depicting the reaction. Cax-Pt was transcribed and then cut by Cla I to form the Cla I-cut EC-400, which was further incubated with different combinations of four repair factors (the omitted

    Journal:

    Article Title: Initiation of DNA repair mediated by a stalled RNA polymerase IIO

    doi: 10.1038/sj.emboj.7600933

    Figure Lengend Snippet: Sequential assembly of NER factors onto RNA pol IIO. ( A ) Scheme depicting the reaction. Cax-Pt was transcribed and then cut by Cla I to form the Cla I-cut EC-400, which was further incubated with different combinations of four repair factors (the omitted

    Article Snippet: Cax-Pt was then cut by Cla I (New England Biolabs) in a reaction volume of 40 ml.

    Techniques: Incubation

    Confirmation of xer1 disruption in selected M . agalactiae ‘switchover’ clones via PCR and Southern analysis. (A) Verification of xer1 disruption in crude DNA extracts of selected ‘switchover’ clones by PCR using primers RecEndET28 and T3ISLrev specific to the chromosomal xer1 region and the pR3 plasmid backbone, respectively. 2 kb PCR product confirms xer1 disruption [ 13 ] in selected ‘switchover’ clones picked from the right (R)—parotideal (PAR) or—mandibular (MAN) lymph nodes of sheep MS 7 or MS 10. The observed bands were comparable to the positive controls corresponding to the crude DNA extracts (PLMY and PLMU) as well to pure DNA preparation of PLMY (+DNA); as expected this band was absent in the three negative controls corresponding to water (H 2 O), PG2 crude extract (PG2) and SP4 broth (Medium). (B) Southerns were performed as described earlier [ 13 ] whereby Cla I-digested genomic DNA was hybridized with xer1 -specific probe. xer1 disruption is confirmed in the ‘switchover’ clones (lanes 3–8) by the presence of two bands of 3.7 kb and ~18.9 kb as also seen for PLMU control (lane 2), whereas the wild type PG2 (lane 1) with intact xer1 shows a 13 kb band. Disruption plasmid pR3 (lane 9) shows the expected 10 kb band. Switchover clones: lane 3, MS 10 right udder/VpmaV; lane 4, MS 6 mesenterial LN/VpmaW; lane 5, MS 6 right iliac LN/VpmaX; lane 6, MS 6 mesenterial LN/VpmaW (PLM W); lane 7, MS 9 right udder/VpmaX (PLM X) ; lane 8, MS 6 right iliac LN/VpmaX.

    Journal: PLoS Pathogens

    Article Title: Vpma phase variation is important for survival and persistence of Mycoplasma agalactiae in the immunocompetent host

    doi: 10.1371/journal.ppat.1006656

    Figure Lengend Snippet: Confirmation of xer1 disruption in selected M . agalactiae ‘switchover’ clones via PCR and Southern analysis. (A) Verification of xer1 disruption in crude DNA extracts of selected ‘switchover’ clones by PCR using primers RecEndET28 and T3ISLrev specific to the chromosomal xer1 region and the pR3 plasmid backbone, respectively. 2 kb PCR product confirms xer1 disruption [ 13 ] in selected ‘switchover’ clones picked from the right (R)—parotideal (PAR) or—mandibular (MAN) lymph nodes of sheep MS 7 or MS 10. The observed bands were comparable to the positive controls corresponding to the crude DNA extracts (PLMY and PLMU) as well to pure DNA preparation of PLMY (+DNA); as expected this band was absent in the three negative controls corresponding to water (H 2 O), PG2 crude extract (PG2) and SP4 broth (Medium). (B) Southerns were performed as described earlier [ 13 ] whereby Cla I-digested genomic DNA was hybridized with xer1 -specific probe. xer1 disruption is confirmed in the ‘switchover’ clones (lanes 3–8) by the presence of two bands of 3.7 kb and ~18.9 kb as also seen for PLMU control (lane 2), whereas the wild type PG2 (lane 1) with intact xer1 shows a 13 kb band. Disruption plasmid pR3 (lane 9) shows the expected 10 kb band. Switchover clones: lane 3, MS 10 right udder/VpmaV; lane 4, MS 6 mesenterial LN/VpmaW; lane 5, MS 6 right iliac LN/VpmaX; lane 6, MS 6 mesenterial LN/VpmaW (PLM W); lane 7, MS 9 right udder/VpmaX (PLM X) ; lane 8, MS 6 right iliac LN/VpmaX.

    Article Snippet: Confirming xer1 disruption via Southern blot analyses Mycoplasma genomic DNA was isolated by QIAamp DNA Mini Kit (Qiagen) and digested with Cla I (New England Biolabs) at 37°C for at least 6–7 h before subjecting to electrophoresis on a 1% agarose gel.

    Techniques: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation, Mass Spectrometry

    Validation of TGR of WT- ply from IDM- ply mutant. (A) Expression of WT-PLY in the 8 h, Day 1 and Day 2 IDM- ply culture by real-time PCR (B) Cla I- digested D39, IDM- ply and AG2, which was sensitive to erm and was obtained from Day 1 culture of IDM- ply mutant, were probed with a digoxigenin-labeled ply-pb in southern blot hybridization. (C) AG2 would produce PLY whose size was the same as PLY of D39 when incubating with a rabbit polyclonal PLY antibody.

    Journal: BMC Biotechnology

    Article Title: Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

    doi: 10.1186/1472-6750-14-16

    Figure Lengend Snippet: Validation of TGR of WT- ply from IDM- ply mutant. (A) Expression of WT-PLY in the 8 h, Day 1 and Day 2 IDM- ply culture by real-time PCR (B) Cla I- digested D39, IDM- ply and AG2, which was sensitive to erm and was obtained from Day 1 culture of IDM- ply mutant, were probed with a digoxigenin-labeled ply-pb in southern blot hybridization. (C) AG2 would produce PLY whose size was the same as PLY of D39 when incubating with a rabbit polyclonal PLY antibody.

    Article Snippet: Kit (Qiagen, Hilden, Germany) and were then digested by Cla I (New England Biolabs, Ipswich, MA, USA) according to the manufactures’ instruction.

    Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Labeling, Southern Blot, Hybridization

    Construction of the ply and lytA knockdown mutants by IDM. Partial sequences of pneumolysin ( ply -s) (A) and autolysin ( lytA -s) (B) genes were cloned into plasmids pVA891 (A) and pEVP3 (B) , containing an erythromycin (erm)- and a chloramphenicol (cat)-resistance marker, respectively. Cloned plasmids were then used to generate homologous recombinants from their WT ply or lytA genes, respectively. Two primer sets, ply-P1/pVA891-R and ply-P2/pVA891-F or lytA-P1/pEVP3-R and lytA-P2/pEVP3-F, were used to verify the insertion of pVA891 and pEVP3 in IDM- ply (A) and - lytA (B) , respectively. C, Cla I.

    Journal: BMC Biotechnology

    Article Title: Differences in virulence of pneumolysin and autolysin mutants constructed by insertion duplication mutagenesis and in-frame deletion in Streptococcus pneumoniae

    doi: 10.1186/1472-6750-14-16

    Figure Lengend Snippet: Construction of the ply and lytA knockdown mutants by IDM. Partial sequences of pneumolysin ( ply -s) (A) and autolysin ( lytA -s) (B) genes were cloned into plasmids pVA891 (A) and pEVP3 (B) , containing an erythromycin (erm)- and a chloramphenicol (cat)-resistance marker, respectively. Cloned plasmids were then used to generate homologous recombinants from their WT ply or lytA genes, respectively. Two primer sets, ply-P1/pVA891-R and ply-P2/pVA891-F or lytA-P1/pEVP3-R and lytA-P2/pEVP3-F, were used to verify the insertion of pVA891 and pEVP3 in IDM- ply (A) and - lytA (B) , respectively. C, Cla I.

    Article Snippet: Kit (Qiagen, Hilden, Germany) and were then digested by Cla I (New England Biolabs, Ipswich, MA, USA) according to the manufactures’ instruction.

    Techniques: Clone Assay, Marker