bsrgi  (New England Biolabs)


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    Structured Review

    New England Biolabs bsrgi
    Bsrgi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsrgi/product/New England Biolabs
    Average 99 stars, based on 50 article reviews
    Price from $9.99 to $1999.99
    bsrgi - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Amplification:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The 998-bp aac(3)-IV (Aprr )- and 569-bp ble (Zeor )-containing fragments were PCR amplified from the pFAM1 and pFZE1 plasmids (primer sequences are provided in ), respectively, and purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek). .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above.

    Agarose Gel Electrophoresis:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The 998-bp aac(3)-IV (Aprr )- and 569-bp ble (Zeor )-containing fragments were PCR amplified from the pFAM1 and pFZE1 plasmids (primer sequences are provided in ), respectively, and purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek). .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The resulting plasmid backbone (5,374-bp fragment) was purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, GA, USA) according to the manufacturer’s protocol.

    Polymerase Chain Reaction:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above. .. The ligated plasmids were transformed into E. coli DH5α, and colonies were selected on LB plus 30 μg/ml Apr (Apr30 ) for pUC18T-mini-Tn 7 T-LAC-Apr transformants or on LB plus 25 μg/ml Zeo (Zeo25 ) for pUC18T-mini-Tn 7 T-LAC-Zeo transformants.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The 998-bp aac(3)-IV (Aprr )- and 569-bp ble (Zeor )-containing fragments were PCR amplified from the pFAM1 and pFZE1 plasmids (primer sequences are provided in ), respectively, and purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek).

    Next-Generation Sequencing:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above. .. Further confirmation was carried out by sequencing both plasmids by Illumina Hi-Seq next-generation sequencing (MGH CCIB DNA Core Facility, Cambridge, MA, USA).

    Purification:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The 998-bp aac(3)-IV (Aprr )- and 569-bp ble (Zeor )-containing fragments were PCR amplified from the pFAM1 and pFZE1 plasmids (primer sequences are provided in ), respectively, and purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek). .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The resulting plasmid backbone (5,374-bp fragment) was purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, GA, USA) according to the manufacturer’s protocol.

    Marker:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: .. The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The resulting plasmid backbone (5,374-bp fragment) was purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, GA, USA) according to the manufacturer’s protocol.

    Sequencing:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above. .. Further confirmation was carried out by sequencing both plasmids by Illumina Hi-Seq next-generation sequencing (MGH CCIB DNA Core Facility, Cambridge, MA, USA).

    Gel Extraction:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The 998-bp aac(3)-IV (Aprr )- and 569-bp ble (Zeor )-containing fragments were PCR amplified from the pFAM1 and pFZE1 plasmids (primer sequences are provided in ), respectively, and purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek). .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The resulting plasmid backbone (5,374-bp fragment) was purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, GA, USA) according to the manufacturer’s protocol.

    Transformation Assay:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above. .. The ligated plasmids were transformed into E. coli DH5α, and colonies were selected on LB plus 30 μg/ml Apr (Apr30 ) for pUC18T-mini-Tn 7 T-LAC-Apr transformants or on LB plus 25 μg/ml Zeo (Zeo25 ) for pUC18T-mini-Tn 7 T-LAC-Zeo transformants.

    Plasmid Preparation:

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: .. PCR products were digested with BsrGI and SacII (New England Biolabs) and the fragments ligated into the mini-Tn 7 plasmid backbone digested with the same enzymes mentioned above. .. The ligated plasmids were transformed into E. coli DH5α, and colonies were selected on LB plus 30 μg/ml Apr (Apr30 ) for pUC18T-mini-Tn 7 T-LAC-Apr transformants or on LB plus 25 μg/ml Zeo (Zeo25 ) for pUC18T-mini-Tn 7 T-LAC-Zeo transformants.

    Article Title: Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
    Article Snippet: .. The Gmr marker from pUC18T-mini-Tn 7 T-LAC-Gm was excised using BsrGI and SacII (New England Biolabs, ON, Canada), leaving the FRT sites in the plasmid backbone intact. .. The resulting plasmid backbone (5,374-bp fragment) was purified from the agarose gel using the E.Z.N.A. gel extraction kit (Omega Bio-Tek, GA, USA) according to the manufacturer’s protocol.

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    New England Biolabs bsr gi
    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 <t>Bsr</t> 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 <t>βGal</t> fragment, which should run slightly slower than the vectors, was not seen in either lane.
    Bsr Gi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsr gi/product/New England Biolabs
    Average 99 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    bsr gi - by Bioz Stars, 2020-04
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    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

    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

    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

    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