bsrgi New England Biolabs Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 96
    New England Biolabs bsrgi
    Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb <t>ASKO.</t> ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with <t>BsrGI</t> and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.
    Bsrgi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsrgi/product/New England Biolabs
    Average 96 stars, based on 616 article reviews
    Price from $9.99 to $1999.99
    bsrgi - by Bioz Stars, 2020-09
    96/100 stars
      Buy from Supplier

    99
    Thermo Fisher bpii bbsi 10 u µl
    Prime editing in cultured S2R+ cells. A. Diagram of PE2 expression plasmid pUAS-PE2 . UAS , Upstream activating sequence ; NLS, Nuclear localization sequence; SV40, 3’ UTR; w+ , white+ rescue transgene; attB , phiC31 recombination site. B. Diagram of <t>pCFD3-NS</t> pegRNA expression plasmid. <t>BbsI</t> sites indicate cloning site for pegRNA encoding sequence. dU6:3 , U6 promoter; U6 3’, U6 downstream region; v+ , vermillion+ rescue transgene. C. ebony genomic region showing target site and installed edit ( ebony G111X ). D. Dual sgRNA and pegRNA expression plasmid pCFD5-NS . tRNA, D.m. and O.s. Gly tRNA sequence. E. Schematic of S2R+ prime editing experiment. F. Quantification of precise editing and indels from S2R+ transfection experiments by amplicon sequencing. tfx, transfection.
    Bpii Bbsi 10 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bpii bbsi 10 u µl/product/Thermo Fisher
    Average 99 stars, based on 23 article reviews
    Price from $9.99 to $1999.99
    bpii bbsi 10 u µl - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 dna ligase
    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with <t>T4</t> DNA ligase.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 49148 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs rnase h
    TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and <t>RNase</t> H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.
    Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h/product/New England Biolabs
    Average 99 stars, based on 4235 article reviews
    Price from $9.99 to $1999.99
    rnase h - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs hindiii
    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: <t>HindIII,</t> EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .
    Hindiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hindiii/product/New England Biolabs
    Average 99 stars, based on 8144 article reviews
    Price from $9.99 to $1999.99
    hindiii - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t4 pnk
    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: <t>HindIII,</t> EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 pnk/product/New England Biolabs
    Average 99 stars, based on 4190 article reviews
    Price from $9.99 to $1999.99
    t4 pnk - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs klenow fragment
    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: <t>HindIII,</t> EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/New England Biolabs
    Average 99 stars, based on 7893 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs ecori
    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: <t>HindIII,</t> <t>EcoRI,</t> BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .
    Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecori/product/New England Biolabs
    Average 99 stars, based on 10938 article reviews
    Price from $9.99 to $1999.99
    ecori - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs xbai
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Xbai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6141 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xbai/product/New England Biolabs
    Average 99 stars, based on 6141 article reviews
    Price from $9.99 to $1999.99
    xbai - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs nhei
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Nhei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nhei/product/New England Biolabs
    Average 99 stars, based on 3250 article reviews
    Price from $9.99 to $1999.99
    nhei - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    95
    New England Biolabs bsrgi hf
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Bsrgi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsrgi hf/product/New England Biolabs
    Average 95 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    bsrgi hf - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    99
    New England Biolabs bamhi
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 11236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bamhi/product/New England Biolabs
    Average 99 stars, based on 11236 article reviews
    Price from $9.99 to $1999.99
    bamhi - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs gibson assembly master mix
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Gibson Assembly Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5227 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gibson assembly master mix/product/New England Biolabs
    Average 99 stars, based on 5227 article reviews
    Price from $9.99 to $1999.99
    gibson assembly master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    91
    Omega Bio-tek e z n a gel extraction kit
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    E Z N A Gel Extraction Kit, supplied by Omega Bio-tek, used in various techniques. Bioz Stars score: 91/100, based on 1547 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e z n a gel extraction kit/product/Omega Bio-tek
    Average 91 stars, based on 1547 article reviews
    Price from $9.99 to $1999.99
    e z n a gel extraction kit - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    99
    New England Biolabs mlui
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Mlui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mlui/product/New England Biolabs
    Average 99 stars, based on 1388 article reviews
    Price from $9.99 to $1999.99
    mlui - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs phusion high fidelity dna polymerase
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Phusion High Fidelity Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 23530 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion high fidelity dna polymerase/product/New England Biolabs
    Average 99 stars, based on 23530 article reviews
    Price from $9.99 to $1999.99
    phusion high fidelity dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs xhoi
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Xhoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/xhoi/product/New England Biolabs
    Average 99 stars, based on 9905 article reviews
    Price from $9.99 to $1999.99
    xhoi - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs alkaline phosphatase
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2053 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alkaline phosphatase/product/New England Biolabs
    Average 99 stars, based on 2053 article reviews
    Price from $9.99 to $1999.99
    alkaline phosphatase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs rna ligase 1
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Rna Ligase 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna ligase 1/product/New England Biolabs
    Average 99 stars, based on 197 article reviews
    Price from $9.99 to $1999.99
    rna ligase 1 - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs antarctic phosphatase
    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites <t>(BsrGI</t> and <t>XbaI)</t> used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.
    Antarctic Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6925 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antarctic phosphatase/product/New England Biolabs
    Average 99 stars, based on 6925 article reviews
    Price from $9.99 to $1999.99
    antarctic phosphatase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs t7 endonuclease i
    Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcriptional activators to cultured human cells. ( a ) Green entries: U2OS EGFP reporter cells were treated with 100 nM of the Cas9 protein variant shown, 0.8 µL of the cationic lipid shown, and either 50 nM of the sgRNA shown for Cas9 protein treatment, or 125 nM of the sgRNA shown for (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 treatment. The fraction of cells lacking EGFP expression was measured by flow cytometry. Blue entries: plasmid DNA transfection of 750 ng Cas9 and 250 ng sgRNA expression plasmids using 0.8 µL Lipofectamine 2000. ( b ) T7 endonuclease I (T7EI) assay to measure modification of EGFP from no treatment (lane 1), treatment with EGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9 protein + VEGF-targeting sgRNA + RNAiMAX (lane 4), DNA transfection of plasmids expressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9 protein + EGFP-targeting sgRNA + RNAiMAX (lane 6). ( c ) T7EI assay of simultaneous genome modification at  EGFP  and three endogenous genes in U2OS cells 48 hours after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA), and 0.8 µL RNAiMAX. ( d ) Delivery of Cas9 D10A nickase and pairs of sgRNAs either by plasmid transfection or by RNAiMAX-mediated protein:sgRNA complex delivery under conditions described in ( a ) with 50 nM  EGFP -disrupting sgRNAs (25 nM each) for protein delivery, and 250 ng sgRNA-expressing plasmids (125 ng each) for DNA delivery.  EGFP -disrupting sgRNAs g1 + g5, or g3 + g7, are expected to result in gene disruption, while g5 + g7 target the same strand and are expected to be non-functional. ( e ) Delivery of dCas9-VP64 transcriptional activators that target  NTF3  either by DNA transfection or RNAiMAX-mediated protein delivery. Error bars reflect s.d. from six biological replicates performed on different days.
    T7 Endonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3560 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 endonuclease i/product/New England Biolabs
    Average 99 stars, based on 3560 article reviews
    Price from $9.99 to $1999.99
    t7 endonuclease i - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs hifi dna assembly master mix
    Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcriptional activators to cultured human cells. ( a ) Green entries: U2OS EGFP reporter cells were treated with 100 nM of the Cas9 protein variant shown, 0.8 µL of the cationic lipid shown, and either 50 nM of the sgRNA shown for Cas9 protein treatment, or 125 nM of the sgRNA shown for (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 treatment. The fraction of cells lacking EGFP expression was measured by flow cytometry. Blue entries: plasmid DNA transfection of 750 ng Cas9 and 250 ng sgRNA expression plasmids using 0.8 µL Lipofectamine 2000. ( b ) T7 endonuclease I (T7EI) assay to measure modification of EGFP from no treatment (lane 1), treatment with EGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9 protein + VEGF-targeting sgRNA + RNAiMAX (lane 4), DNA transfection of plasmids expressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9 protein + EGFP-targeting sgRNA + RNAiMAX (lane 6). ( c ) T7EI assay of simultaneous genome modification at  EGFP  and three endogenous genes in U2OS cells 48 hours after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA), and 0.8 µL RNAiMAX. ( d ) Delivery of Cas9 D10A nickase and pairs of sgRNAs either by plasmid transfection or by RNAiMAX-mediated protein:sgRNA complex delivery under conditions described in ( a ) with 50 nM  EGFP -disrupting sgRNAs (25 nM each) for protein delivery, and 250 ng sgRNA-expressing plasmids (125 ng each) for DNA delivery.  EGFP -disrupting sgRNAs g1 + g5, or g3 + g7, are expected to result in gene disruption, while g5 + g7 target the same strand and are expected to be non-functional. ( e ) Delivery of dCas9-VP64 transcriptional activators that target  NTF3  either by DNA transfection or RNAiMAX-mediated protein delivery. Error bars reflect s.d. from six biological replicates performed on different days.
    Hifi Dna Assembly Master Mix, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hifi dna assembly master mix/product/New England Biolabs
    Average 99 stars, based on 1304 article reviews
    Price from $9.99 to $1999.99
    hifi dna assembly master mix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb ASKO. ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with BsrGI and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.

    Journal: Nature Communications

    Article Title: Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections

    doi: 10.1038/ncomms9775

    Figure Lengend Snippet: Generation of AS knockout in P. berghei . ( a ) Double cross-over recombination strategy followed to generate Pb ASKO. ( b ) PCR analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: PCR with Pb AS-specific primers; Lane 2 and 4: PCR with Pb GAPDH-specific primers (control); Lane M: 1 kb DNA ladder. ( c ) RT-PCR analysis for RNA isolated from Pb WT and Pb ASKO parasites. Lane 1 and 3: RT-PCR with Pb AS-specific primers; Lane 2 and 4: RT-PCR with Pb GAPDH specific primers (control); Lane M: 1 kb DNA ladder. ( d ) Southern analysis for genomic DNA isolated from Pb WT and Pb ASKO parasites. Transgenic plasmid (TP) was also included as control to rule out the presence of any episomes. Genomic DNA preparations and TP were digested with BsrGI and XbaI followed by hybridization with 5′-UTR specific probe. ( e ) Northern analysis for RNA isolated from Pb WT and Pb ASKO parasites indicating the absence of AS mRNA (1.75 kb) in Pb ASKO. For control, GAPDH (1.01 kb; lower panel) was used (full blots are shown in Supplementary Fig. 6 ). ( f ) Western blot analysis indicating the absence of AS (67 kDa) in Pb ASKO parasite lysate ( Supplementary Fig. 6 ). Parasite lysates of Pb WT and Pb ASKO containing 100 μg of total protein were used. For control, hsp60 (60 kDa) was used (lower panel). ( g ) Enzyme assays for Pb WT and Pb ASKO parasites using [U- 14 C]-aspartate. 200 μg of total protein was used per assay.

    Article Snippet: For Southern analysis, genomic DNA preparations (10 μg) from Pb WT and Pb ASKO parasites were subjected to BsrGI and XbaI digestion followed by hybridization with 5′-UTR-specific probe that was synthesized using Klenow Fragment (New England Biolabs) with 5′-UTR PCR product as a template in the presence of 5 μCi [α-32 P]-dATP.

    Techniques: Knock-Out, Polymerase Chain Reaction, Isolation, Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Plasmid Preparation, Hybridization, Northern Blot, Western Blot

    Comparison of cloned plasmids produced from an equimolar mixture of three DNA fragments of different sizes by RC and LC. Plasmids retrieved from transformants generated by RC and LC were digested with Bgl II ( A ) or Bsr GI ( B ), and then run on 0.7 or 1.2% agarose gels, respectively. Plasmids were linearized by Bgl II digestion, and inserts were excised from the vector (pSP73) by Bsr GI digestion. Because the resultant clones by RC contained recombination site sequences in the vector portion, the size of the RC plasmid became slightly larger than that of the LC plasmid even when it carried the same insert. In (B), contaminating E.coli chromosomal DNA appeared as the uppermost band in both lanes and the excised βGal fragment, which should run slightly slower than the vectors, was not seen in either lane.

    Journal: Nucleic Acids Research

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction

    doi:

    Figure Lengend Snippet: Comparison of cloned plasmids produced from an equimolar mixture of three DNA fragments of different sizes by RC and LC. Plasmids retrieved from transformants generated by RC and LC were digested with Bgl II ( A ) or Bsr GI ( B ), and then run on 0.7 or 1.2% agarose gels, respectively. Plasmids were linearized by Bgl II digestion, and inserts were excised from the vector (pSP73) by Bsr GI digestion. Because the resultant clones by RC contained recombination site sequences in the vector portion, the size of the RC plasmid became slightly larger than that of the LC plasmid even when it carried the same insert. In (B), contaminating E.coli chromosomal DNA appeared as the uppermost band in both lanes and the excised βGal fragment, which should run slightly slower than the vectors, was not seen in either lane.

    Article Snippet: MCS, Tet and βGal thus obtained were digested with Bsr GI (New England BioLabs), which cleaves at the TGTACA sequence within the common 15 bp core region of each att sequence, leaving four-base 5′ overhangs.

    Techniques: Clone Assay, Produced, Generated, Plasmid Preparation

    Assay of potential Tre-related off-target effects. (A) Nucleotide sequences of genomic sites and their locations in the human genome (in brackets). Sequences are aligned to the Tre recognition site loxLTR. Nucleotides that differ from loxLTR are shown in red. (B) Representation of the recombination assay in E. coli . and in HeLa cells, respectively. The evolution vector pEVO-Tre-target contains two directly repeated recombinase target sites (loxLTR) or the sequences GS1, GS2, GS3, GS4, GS5, GS6, and GS7. In E. coli , Tre is expressed from the P BAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC, and a chloramphenicol resistance marker (Cm r ). Recombination at the target sites leads to deletion of the 700 bp intervening region. Locations of the PCR primer binding sites (F, R) for detection of recombination are indicated. (C) Agarose gel showing the activity of Tre on loxLTR and the lack thereof for the seven genomic sites GS1 to GS7 (lanes 3–9). Upper panel: Recombination assayed in E. Coli . BsrG I/Xba I restriction digest results in a 4.9 kb fragment for non-recombined plasmid (two triangles) and a 4.2 kb fragment for recombined product (one triangle). Recombination tests on loxLTR served as negative and positive control (lanes 1 and 2). −, non-induced; +, induced with 1 mg/ml L-arabinose; M, DNA marker lane. Lower panel: Recombination assayed in HeLa cells. PCR using primers F and R that anneal to the vector DNA results in a 0.4 kb product when recombination occurs, while the non-recombined template results in a 1.1 kb PCR product. −, cotransfection with pIRESneo (i.e. no Tre expression); +, cotransfection with pIRESneo-Tre [18] .

    Journal: PLoS Pathogens

    Article Title: Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice

    doi: 10.1371/journal.ppat.1003587

    Figure Lengend Snippet: Assay of potential Tre-related off-target effects. (A) Nucleotide sequences of genomic sites and their locations in the human genome (in brackets). Sequences are aligned to the Tre recognition site loxLTR. Nucleotides that differ from loxLTR are shown in red. (B) Representation of the recombination assay in E. coli . and in HeLa cells, respectively. The evolution vector pEVO-Tre-target contains two directly repeated recombinase target sites (loxLTR) or the sequences GS1, GS2, GS3, GS4, GS5, GS6, and GS7. In E. coli , Tre is expressed from the P BAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC, and a chloramphenicol resistance marker (Cm r ). Recombination at the target sites leads to deletion of the 700 bp intervening region. Locations of the PCR primer binding sites (F, R) for detection of recombination are indicated. (C) Agarose gel showing the activity of Tre on loxLTR and the lack thereof for the seven genomic sites GS1 to GS7 (lanes 3–9). Upper panel: Recombination assayed in E. Coli . BsrG I/Xba I restriction digest results in a 4.9 kb fragment for non-recombined plasmid (two triangles) and a 4.2 kb fragment for recombined product (one triangle). Recombination tests on loxLTR served as negative and positive control (lanes 1 and 2). −, non-induced; +, induced with 1 mg/ml L-arabinose; M, DNA marker lane. Lower panel: Recombination assayed in HeLa cells. PCR using primers F and R that anneal to the vector DNA results in a 0.4 kb product when recombination occurs, while the non-recombined template results in a 1.1 kb PCR product. −, cotransfection with pIRESneo (i.e. no Tre expression); +, cotransfection with pIRESneo-Tre [18] .

    Article Snippet: Plasmid DNA was isolated from overnight cultures and digested with BsrG I and Xba I (NEB), resulting in different fragment sizes for recombined versus non-recombined substrate on agarose gels.

    Techniques: Recombination Assay, Plasmid Preparation, Marker, Polymerase Chain Reaction, Binding Assay, Agarose Gel Electrophoresis, Activity Assay, Positive Control, Cotransfection, Expressing

    Prime editing in cultured S2R+ cells. A. Diagram of PE2 expression plasmid pUAS-PE2 . UAS , Upstream activating sequence ; NLS, Nuclear localization sequence; SV40, 3’ UTR; w+ , white+ rescue transgene; attB , phiC31 recombination site. B. Diagram of pCFD3-NS pegRNA expression plasmid. BbsI sites indicate cloning site for pegRNA encoding sequence. dU6:3 , U6 promoter; U6 3’, U6 downstream region; v+ , vermillion+ rescue transgene. C. ebony genomic region showing target site and installed edit ( ebony G111X ). D. Dual sgRNA and pegRNA expression plasmid pCFD5-NS . tRNA, D.m. and O.s. Gly tRNA sequence. E. Schematic of S2R+ prime editing experiment. F. Quantification of precise editing and indels from S2R+ transfection experiments by amplicon sequencing. tfx, transfection.

    Journal: bioRxiv

    Article Title: Precise genome engineering in Drosophila using prime editing

    doi: 10.1101/2020.08.05.232348

    Figure Lengend Snippet: Prime editing in cultured S2R+ cells. A. Diagram of PE2 expression plasmid pUAS-PE2 . UAS , Upstream activating sequence ; NLS, Nuclear localization sequence; SV40, 3’ UTR; w+ , white+ rescue transgene; attB , phiC31 recombination site. B. Diagram of pCFD3-NS pegRNA expression plasmid. BbsI sites indicate cloning site for pegRNA encoding sequence. dU6:3 , U6 promoter; U6 3’, U6 downstream region; v+ , vermillion+ rescue transgene. C. ebony genomic region showing target site and installed edit ( ebony G111X ). D. Dual sgRNA and pegRNA expression plasmid pCFD5-NS . tRNA, D.m. and O.s. Gly tRNA sequence. E. Schematic of S2R+ prime editing experiment. F. Quantification of precise editing and indels from S2R+ transfection experiments by amplicon sequencing. tfx, transfection.

    Article Snippet: pCFD3-NS (Addgene #149545) pCFD3 (Addgene #49410) ( ) was digested with BbsI (Fermentas ER1011) and XbaI (New England Biolabs R0145), which removes the sgRNA scaffold and Drosophila U6 downstream region, and the backbone was purified using a QIAquick column (28115, Qiagen).

    Techniques: Cell Culture, Expressing, Plasmid Preparation, Sequencing, Clone Assay, Transfection, Amplification

    Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.

    Journal: Biological Procedures Online

    Article Title: An Efficient Ligation Method in the Making of an in vitro Virus for in vitro Protein Evolution

    doi: 10.1251/bpo33

    Figure Lengend Snippet: Gel-electrophoresis of ligation products. The ligation products were electrophoresed on 8M Urea 10% PAGE at 65 ºC and were visualized with fluorescence of FITC and then visualized after staining by SYBR Green II using an imager. Lane CY: control, Linker-Y. Lane CS: control, mRNA without ligation. Lane CR: control, Linker-S. Lane Y: Y-ligation with T4 RNA ligase. Lane R: splint ligation with T4 RNA ligase. Lane D: splint ligation with T4 DNA ligase.

    Article Snippet: The ligation reaction with T4 DNA ligase (10 U/µl, NEB) was performed according to the supplier's recommendation at 25 ºC.

    Techniques: Nucleic Acid Electrophoresis, Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Staining, SYBR Green Assay

    TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.

    Journal: Molecular Cell

    Article Title: Elongation Factor TFIIS Prevents Transcription Stress and R-Loop Accumulation to Maintain Genome Stability

    doi: 10.1016/j.molcel.2019.07.037

    Figure Lengend Snippet: TFIIS mut Expression Results in an Accumulation of R-Loops (A) RNA or DNA hybrid slot-blot of genomic DNA from TFIIS mut and parental cells, ±RNase H. S9.6 antibody was used to detect RNA or DNA hybrids (upper panel on right) with single-strand DNA antibody (bottom panel) as a loading control. Serial dilutions of genomic DNA (1/1 = 4 μg) were probed with S9.6 antibody for standards (left panel). (B) Fold enrichment in RNA or DNA hybrids compared with control (n = 3). Mean ± SEM (bars) values are shown. p values were determined by unpaired t test. (C and D) DRIP-qPCR analysis of R-loop induction at the SOX4 gene (C) and the SNRPN gene (D) (n = 3). Mean ± SEM (bars) values are shown. p values were determined by two-way ANOVA statistical test. (E) Left: schematic of idealized experiment. Radioactive label is denoted by red dot and the biotin tag on DNA with a black dot. The position of the first adenine in the transcript is also indicated. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Ambion RNA size markers are indicated on the left for approximate RNA sizes. Positions of full-length product (FL), R-loops, and cleavage products are indicated on the right. (F) Left: experimental scheme; similar to that of (E) but involving purification via the biotin tag after RNase H digestion. Right: R-loop detection by denaturing PAGE after addition of TFIIS proteins and RNase H to yeast TECs assembled in vitro . Approximate RNA sizes RNA and position of R-loops are indicated on the right and next to relevant lanes. Asterisk-bar denotes irrelevant pausing sites of unknown origin, including IC1 and IC2. See Figure S5 for detailed schematic explanations.

    Article Snippet: Half of the sample was digested with 40U RNase H (NEB) to serve as negative control, for 24 hours at 37°C.

    Techniques: Expressing, Dot Blot, Real-time Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, In Vitro, Purification

    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .

    Journal: Genome Research

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

    doi: 10.1101/gr.219394.116

    Figure Lengend Snippet: Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .

    Article Snippet: 2, 4, 6, 8, 10, 12, 14, and 16, purified DNA samples (∼25 µg each) were fragmented using a restriction enzyme cocktail of 1 µL HindIII (20 U/µL), 1 µL EcoRI (20 U/µL), 2 µL BsrGI (10 U/µL), 1 µL XbaI (20 U/µL), and 4 µL SspI (5 U/µL) in NEB Buffer 2 (NEB) (V = 300 µL) at 37°C for 4 h. The fragmented DNA samples were repurified either by phenol-chloroform extraction (experiments 1–4, 9–12) or by the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) (experiments 5–8, 13–16).

    Techniques:

    Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .

    Journal: Genome Research

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

    doi: 10.1101/gr.219394.116

    Figure Lengend Snippet: Analysis of restriction sites over genic and intergenic regions. ( A ) Restriction fragment lengths over genic regions (gene bodies, exons, first exons) are significantly larger compared to intergenic regions. The plot shows the difference of genic (observed) and intergenic (expected) fragment sizes in base pairs. The following enzymes were applied in combination: HindIII, EcoRI, BsrGI, XbaI, and SspI. ( B – D ) The number of restriction sites over genic regions is significantly lower compared to intergenic regions. Colors indicate the proportion of cutting sites in each category. Red and blue slices, marking the rarest restriction site frequencies, are prevalent over genic elements in each pie chart. ( E ) Cutting efficiency of restriction enzymes applied in the indicated DRIP-seq experiments. Zero read: the restriction site was cut. Greater equal than one read: the restriction site was uncut in a fraction of cells. There were uncut reads (sites) over half of the theoretical restriction sites. The proportion of uncut reads was even higher within gene coding regions compared to intergenic regions. See the model of cutting efficiency in panel F .

    Article Snippet: 2, 4, 6, 8, 10, 12, 14, and 16, purified DNA samples (∼25 µg each) were fragmented using a restriction enzyme cocktail of 1 µL HindIII (20 U/µL), 1 µL EcoRI (20 U/µL), 2 µL BsrGI (10 U/µL), 1 µL XbaI (20 U/µL), and 4 µL SspI (5 U/µL) in NEB Buffer 2 (NEB) (V = 300 µL) at 37°C for 4 h. The fragmented DNA samples were repurified either by phenol-chloroform extraction (experiments 1–4, 9–12) or by the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) (experiments 5–8, 13–16).

    Techniques:

    Experimental design: constructing DRIP schemes. ( A ) Experiments 1–16 explore the effect of formaldehyde-fixation (Step 1), nucleic acid isolation (Step 2), removal of free RNA (Step 3), and nucleic acid fragmentation (Step 4) on the outcome of RNA-DNA hybrid detection. Each experiment was performed at two parallel cell lysis temperatures (65°C and 37°C), respectively. The temperature variable is not depicted in the cartoon, but it is referred in the main text. ( B ) Experiments 17–24 test the impact of acoustic sharing performed on a chromatin prep rather than on naked nucleic acid, similarly to the ChIP protocol. Each experiment was performed at 65°C cell lysis temperature. ( C ) Workflow of a ChIP experiment (shown only for comparison with the DRIP pipeline). (HCHO) Formaldehyde fixation, (Phe/Chl) phenol-chloroform extraction, (Kit) silica membrane-based nucleic acid purification, (RNase A) Ribonuclease A digestion performed at high (300 mM) NaCl concentration, (Son) sonication, (RE) restriction enzyme cocktail digestion (HindIII, EcoRI, BsrGI, XbaI, and SspI). As a negative control, RNase H digestion was applied in all DRIP experiments (not indicated in the cartoon).

    Journal: Genome Research

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

    doi: 10.1101/gr.219394.116

    Figure Lengend Snippet: Experimental design: constructing DRIP schemes. ( A ) Experiments 1–16 explore the effect of formaldehyde-fixation (Step 1), nucleic acid isolation (Step 2), removal of free RNA (Step 3), and nucleic acid fragmentation (Step 4) on the outcome of RNA-DNA hybrid detection. Each experiment was performed at two parallel cell lysis temperatures (65°C and 37°C), respectively. The temperature variable is not depicted in the cartoon, but it is referred in the main text. ( B ) Experiments 17–24 test the impact of acoustic sharing performed on a chromatin prep rather than on naked nucleic acid, similarly to the ChIP protocol. Each experiment was performed at 65°C cell lysis temperature. ( C ) Workflow of a ChIP experiment (shown only for comparison with the DRIP pipeline). (HCHO) Formaldehyde fixation, (Phe/Chl) phenol-chloroform extraction, (Kit) silica membrane-based nucleic acid purification, (RNase A) Ribonuclease A digestion performed at high (300 mM) NaCl concentration, (Son) sonication, (RE) restriction enzyme cocktail digestion (HindIII, EcoRI, BsrGI, XbaI, and SspI). As a negative control, RNase H digestion was applied in all DRIP experiments (not indicated in the cartoon).

    Article Snippet: 2, 4, 6, 8, 10, 12, 14, and 16, purified DNA samples (∼25 µg each) were fragmented using a restriction enzyme cocktail of 1 µL HindIII (20 U/µL), 1 µL EcoRI (20 U/µL), 2 µL BsrGI (10 U/µL), 1 µL XbaI (20 U/µL), and 4 µL SspI (5 U/µL) in NEB Buffer 2 (NEB) (V = 300 µL) at 37°C for 4 h. The fragmented DNA samples were repurified either by phenol-chloroform extraction (experiments 1–4, 9–12) or by the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) (experiments 5–8, 13–16).

    Techniques: Isolation, Lysis, Chromatin Immunoprecipitation, Nucleic Acid Purification, Concentration Assay, Sonication, Negative Control

    Large restriction fragments over gene bodies cause uncertainty in the precise localization of R-loops, potentially impeding their functional annotation. ( A – C ) Genome browser tracks showing three representative examples ( MYC , BCL6 , and VIM ). Upper two tracks: restriction fragment-sized R-loops are prevalent over the 5′ end of genes, vastly exceeding the gene borders in the case of MYC . Lower two tracks: the precise genomic position of R-loops was resolved in the sonicated group of samples. Green boxes represent R-loop enriched regions predicted by the peak callers. Blue dashed lines represent cutting sites for restriction enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI).

    Journal: Genome Research

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases

    doi: 10.1101/gr.219394.116

    Figure Lengend Snippet: Large restriction fragments over gene bodies cause uncertainty in the precise localization of R-loops, potentially impeding their functional annotation. ( A – C ) Genome browser tracks showing three representative examples ( MYC , BCL6 , and VIM ). Upper two tracks: restriction fragment-sized R-loops are prevalent over the 5′ end of genes, vastly exceeding the gene borders in the case of MYC . Lower two tracks: the precise genomic position of R-loops was resolved in the sonicated group of samples. Green boxes represent R-loop enriched regions predicted by the peak callers. Blue dashed lines represent cutting sites for restriction enzymes (HindIII, EcoRI, BsrGI, XbaI, and SspI).

    Article Snippet: 2, 4, 6, 8, 10, 12, 14, and 16, purified DNA samples (∼25 µg each) were fragmented using a restriction enzyme cocktail of 1 µL HindIII (20 U/µL), 1 µL EcoRI (20 U/µL), 2 µL BsrGI (10 U/µL), 1 µL XbaI (20 U/µL), and 4 µL SspI (5 U/µL) in NEB Buffer 2 (NEB) (V = 300 µL) at 37°C for 4 h. The fragmented DNA samples were repurified either by phenol-chloroform extraction (experiments 1–4, 9–12) or by the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) (experiments 5–8, 13–16).

    Techniques: Functional Assay, Sonication

    Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites (BsrGI and XbaI) used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.

    Journal: Scientific Reports

    Article Title: Nearest-neighbor amino acids of specificity-determining residues influence the activity of engineered Cre-type recombinases

    doi: 10.1038/s41598-020-70867-5

    Figure Lengend Snippet: Rationally designed Cre mutants show increased recombination activity in E.coli . ( a ) Schematic drawing of the plasmid assay. Important regions in the plasmids are indicated. Note the reduced size of the plasmid after recombination. The restrictions sites (BsrGI and XbaI) used for cloning indicated Cre-recombinase variants are depicted. The rox target sites are shown as red triangles. CmR, chloramphenicol resistance gene; ori, origin of replication; AraC, arabinose operon regulatory gene. ( b ) Agarose gels of three independently picked clones showing BsrGI and XbaI digested plasmids carrying indicated recombinases. The amount of arabinose added to the growth medium is presented below each band in μg/ml l +-arabinose. The line with two triangles indicates no recombination, whereas the line with one triangle marks the recombined band. Quantifications of the ratios of band intensities (in percent of recombination) are shown to the right for each mutant. The amount of arabinose added to the growth medium is shown on the X-axis. Error bars depict standard deviation from the three independent experiments.

    Article Snippet: Recombination assays in E. coli For expression in E. coli , mCreK and variants thereof were cloned into the pEVOrox vector utilizing the unique BsrGI and XbaI (NEB, Ipswich, MA, USA) restriction sites.

    Techniques: Activity Assay, Plasmid Preparation, Clone Assay, Mutagenesis, Standard Deviation

    Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcriptional activators to cultured human cells. ( a ) Green entries: U2OS EGFP reporter cells were treated with 100 nM of the Cas9 protein variant shown, 0.8 µL of the cationic lipid shown, and either 50 nM of the sgRNA shown for Cas9 protein treatment, or 125 nM of the sgRNA shown for (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 treatment. The fraction of cells lacking EGFP expression was measured by flow cytometry. Blue entries: plasmid DNA transfection of 750 ng Cas9 and 250 ng sgRNA expression plasmids using 0.8 µL Lipofectamine 2000. ( b ) T7 endonuclease I (T7EI) assay to measure modification of EGFP from no treatment (lane 1), treatment with EGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9 protein + VEGF-targeting sgRNA + RNAiMAX (lane 4), DNA transfection of plasmids expressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9 protein + EGFP-targeting sgRNA + RNAiMAX (lane 6). ( c ) T7EI assay of simultaneous genome modification at  EGFP  and three endogenous genes in U2OS cells 48 hours after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA), and 0.8 µL RNAiMAX. ( d ) Delivery of Cas9 D10A nickase and pairs of sgRNAs either by plasmid transfection or by RNAiMAX-mediated protein:sgRNA complex delivery under conditions described in ( a ) with 50 nM  EGFP -disrupting sgRNAs (25 nM each) for protein delivery, and 250 ng sgRNA-expressing plasmids (125 ng each) for DNA delivery.  EGFP -disrupting sgRNAs g1 + g5, or g3 + g7, are expected to result in gene disruption, while g5 + g7 target the same strand and are expected to be non-functional. ( e ) Delivery of dCas9-VP64 transcriptional activators that target  NTF3  either by DNA transfection or RNAiMAX-mediated protein delivery. Error bars reflect s.d. from six biological replicates performed on different days.

    Journal: Nature biotechnology

    Article Title: Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

    doi: 10.1038/nbt.3081

    Figure Lengend Snippet: Delivery of Cas9:sgRNA, Cas9 D10A nickase, and dCas9-VP64 transcriptional activators to cultured human cells. ( a ) Green entries: U2OS EGFP reporter cells were treated with 100 nM of the Cas9 protein variant shown, 0.8 µL of the cationic lipid shown, and either 50 nM of the sgRNA shown for Cas9 protein treatment, or 125 nM of the sgRNA shown for (+36)dGFP-NLS-Cas9 and (−30)dGFP-NLS-Cas9 treatment. The fraction of cells lacking EGFP expression was measured by flow cytometry. Blue entries: plasmid DNA transfection of 750 ng Cas9 and 250 ng sgRNA expression plasmids using 0.8 µL Lipofectamine 2000. ( b ) T7 endonuclease I (T7EI) assay to measure modification of EGFP from no treatment (lane 1), treatment with EGFP-targeting sgRNA alone (lane 2), Cas9 protein alone (lane 3), Cas9 protein + VEGF-targeting sgRNA + RNAiMAX (lane 4), DNA transfection of plasmids expressing Cas9 and EGFP-targeting sgRNA (lane 5), or Cas9 protein + EGFP-targeting sgRNA + RNAiMAX (lane 6). ( c ) T7EI assay of simultaneous genome modification at EGFP and three endogenous genes in U2OS cells 48 hours after a single treatment of 100 nM Cas9 protein, 25 nM of each of the four sgRNAs shown (100 nM total sgRNA), and 0.8 µL RNAiMAX. ( d ) Delivery of Cas9 D10A nickase and pairs of sgRNAs either by plasmid transfection or by RNAiMAX-mediated protein:sgRNA complex delivery under conditions described in ( a ) with 50 nM EGFP -disrupting sgRNAs (25 nM each) for protein delivery, and 250 ng sgRNA-expressing plasmids (125 ng each) for DNA delivery. EGFP -disrupting sgRNAs g1 + g5, or g3 + g7, are expected to result in gene disruption, while g5 + g7 target the same strand and are expected to be non-functional. ( e ) Delivery of dCas9-VP64 transcriptional activators that target NTF3 either by DNA transfection or RNAiMAX-mediated protein delivery. Error bars reflect s.d. from six biological replicates performed on different days.

    Article Snippet: The re-annealed DNA was incubated with 1 µl of T7 Endonuclease I (10 U/µl, NEB) at 37 °C for 15 min. 10 µL of 50 % glycerol was added to the T7 Endonuclease reaction and 12 µL was analyzed on a 5 % TBE 18-well Criterion PAGE gel (Bio-Rad) electrophoresed for 30 min at 200 V, then stained with 1x SYBR Gold (Life Technologies) for 30 min. Cas9-induced cleavage bands and the uncleaved band were visualized on an AlphaImager HP (Alpha Innotech) and quantified using ImageJ software .

    Techniques: Cell Culture, Variant Assay, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, T7EI Assay, Modification, Functional Assay