bsrgi  (New England Biolabs)


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    Name:
    BsrGI
    Description:
    BsrGI 5 000 units
    Catalog Number:
    R0575L
    Price:
    282
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    Structured Review

    New England Biolabs bsrgi
    BsrGI
    BsrGI 5 000 units
    https://www.bioz.com/result/bsrgi/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsrgi - by Bioz Stars, 2021-06
    97/100 stars

    Images

    1) Product Images from "Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections"

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

    Journal: Nature Communications

    doi: 10.1038/ncomms9775

    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.
    Figure Legend 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.

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

    2) Product Images from "RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases"

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

    Journal: Genome Research

    doi: 10.1101/gr.219394.116

    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 .
    Figure Legend 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 .

    Techniques Used:

    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).
    Figure Legend 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).

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: Functional Assay, Sonication

    Related Articles

    Amplification:

    Article Title: Spread of Cryptococcus gattii in British Columbia, Canada, and Detection in the Pacific Northwest, USA
    Article Snippet: Molecular types were identified by a previously described PCR-based restriction fragment length polymorphism (RFLP) method , which was adapted for further discrimination of variation within the VGII molecular type ( ). .. The URA5 gene was amplified as previously described ( ) and then completely digested at 37°C in a 20-μL reaction containing 1× NEB2 buffer, 1× bovine serum albumin, and 4 U each of Hha I, Dde I, and BsrG I (New England Biolabs, Inc., Ipswich, MA, USA). .. RFLP products were subjected to electrophoresis and visualized on a 3% agarose gel prestained with ethidium bromide.

    Plasmid Preparation:

    Article Title: Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice
    Article Snippet: In E. coli , recombinase expression was induced with L-arabinose (Sigma-Aldrich) at 1 mg/ml. .. 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. .. Recombination on the Tre target loxLTR served as positive control.

    Article Title: Human Artificial Chromosomes that Bypass Centromeric DNA
    Article Snippet: Plasmid construction The doxycycline-inducible mCherry-LacI-HJURP donor plasmid (used to make the HT1080Dox-inducible mCherry-LacI-HJURP cell line via RMCE) was constructed by digesting a plasmid containing mCherry-LacI-HJURP ( ) (a gift from D. Foltz, Northwestern University) with PsiI and AgeI-HF to isolate the mCherry-LacI-HJURP gene. .. A donor plasmid containing a TRE floxed by loxP and lox2272 recombination sites ( ) (pEM791; a gift from E. Makeyev, Nanyang Technological University, Singapore) was digested with BsrGI, blunted with Klenow DNA polymerase (NEB), and then digested with AgeI-HF. .. The mCherry-LacI-HJURP fragment was ligated to the pEM791 backbone to produce the doxycycline-inducible mCherry-LacI-HJURP donor plasmid.

    Isolation:

    Article Title: Highly Significant Antiviral Activity of HIV-1 LTR-Specific Tre-Recombinase in Humanized Mice
    Article Snippet: In E. coli , recombinase expression was induced with L-arabinose (Sigma-Aldrich) at 1 mg/ml. .. 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. .. Recombination on the Tre target loxLTR served as positive control.

    Construct:

    Article Title: Identification of a Linear Epitope in Sortilin That Partakes in Pro-neurotrophin Binding *
    Article Snippet: Multiple rounds of mutagenesis allowed for generation of double, triple, and quadruple mutant constructs knocking out several ligand binding residues in the sortilin domain. .. To generate the quadruple mutations in a sortilin construct encoding the entire sortilin receptor, including the transmembrane and cytoplasmic domains, we took advantage of internal HpaI and BsrGI (New England Biolabs catalog nos. .. R0105S and R0575S, respectively) restriction recognition sites in the sortilin sequence.

    Purification:

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases
    Article Snippet: In experiments 1, 3, 5, 7, 9, 11, 13, 15, the purified nucleic acid preps were sonicated in a buffer of 10 mM Tris-HCl pH 8.5 supplemented with 300 mM NaCl (V = 300 µL) for 2 × 5 min (30 sec ON, 30 sec OFF, LOW; Bioruptor, Diagenode) to yield an average DNA fragment size of ∼300 bp. .. 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). ..

    Polymerase Chain Reaction:

    Article Title: RNA-DNA hybrid (R-loop) immunoprecipitation mapping: an analytical workflow to evaluate inherent biases
    Article Snippet: In experiments 1, 3, 5, 7, 9, 11, 13, 15, the purified nucleic acid preps were sonicated in a buffer of 10 mM Tris-HCl pH 8.5 supplemented with 300 mM NaCl (V = 300 µL) for 2 × 5 min (30 sec ON, 30 sec OFF, LOW; Bioruptor, Diagenode) to yield an average DNA fragment size of ∼300 bp. .. 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). ..

    Article Title: Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections
    Article Snippet: Gene deletion in the resulting KO (ASKO) parasites was examined by PCR using PbAS -specific primers and confirmed further by Southern, northern and western analyses. .. 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. .. Northern analysis was carried out with 2 μg of total RNA prepared from Pb WT and Pb ASKO parasites that were hybridized with Pb AS-specific probe synthesized by Klenow Fragment using PbAS cDNA as a template.

    Hybridization:

    Article Title: Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections
    Article Snippet: Gene deletion in the resulting KO (ASKO) parasites was examined by PCR using PbAS -specific primers and confirmed further by Southern, northern and western analyses. .. 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. .. Northern analysis was carried out with 2 μg of total RNA prepared from Pb WT and Pb ASKO parasites that were hybridized with Pb AS-specific probe synthesized by Klenow Fragment using PbAS cDNA as a template.

    Synthesized:

    Article Title: Asparagine requirement in Plasmodium berghei as a target to prevent malaria transmission and liver infections
    Article Snippet: Gene deletion in the resulting KO (ASKO) parasites was examined by PCR using PbAS -specific primers and confirmed further by Southern, northern and western analyses. .. 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. .. Northern analysis was carried out with 2 μg of total RNA prepared from Pb WT and Pb ASKO parasites that were hybridized with Pb AS-specific probe synthesized by Klenow Fragment using PbAS cDNA as a template.

    Sequencing:

    Article Title: Directional cDNA library construction assisted by the in vitro recombination reaction
    Article Snippet: DNA fragments containing the tetracycline-resistance gene (Tet) and beta-galactosidase gene (βGal) were amplified from control plasmids included in the field test kit provided by Life Technologies using the same set of PCR primers. .. 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. .. The products with or without Bsr GI digestion were finally purified using the Concert Rapid PCR Purification System (Life Technologies) and then used for LC or RC, respectively.

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    New England Biolabs bsrg i
    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, <t>DNA</t> 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] .
    Bsrg I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    URA5–restriction fragment length polymorphism (RFLP) profiles for selected human, animal, and environmental Cryptococcus gattii isolates. A) URA5-RFLP to determine the molecular type using Hha I and Sau96 I endonucleases ( 14 ). B) URA5-RFLP to confirm molecular type and determine VGII subtype, using Hha I, Dde I, and BsrG I endonucleases.

    Journal: Emerging Infectious Diseases

    Article Title: Spread of Cryptococcus gattii in British Columbia, Canada, and Detection in the Pacific Northwest, USA

    doi: 10.3201/eid1301.060827

    Figure Lengend Snippet: URA5–restriction fragment length polymorphism (RFLP) profiles for selected human, animal, and environmental Cryptococcus gattii isolates. A) URA5-RFLP to determine the molecular type using Hha I and Sau96 I endonucleases ( 14 ). B) URA5-RFLP to confirm molecular type and determine VGII subtype, using Hha I, Dde I, and BsrG I endonucleases.

    Article Snippet: The URA5 gene was amplified as previously described ( ) and then completely digested at 37°C in a 20-μL reaction containing 1× NEB2 buffer, 1× bovine serum albumin, and 4 U each of Hha I, Dde I, and BsrG I (New England Biolabs, Inc., Ipswich, MA, USA).

    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