drai  (New England Biolabs)


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    Name:
    DraI
    Description:
    DraI 10 000 units
    Catalog Number:
    R0129L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    10 000 units
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    New England Biolabs drai
    DraI
    DraI 10 000 units
    https://www.bioz.com/result/drai/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    drai - by Bioz Stars, 2021-06
    96/100 stars

    Images

    1) Product Images from "Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1"

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku419

    Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. ( A ) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. ( B ) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.
    Figure Legend Snippet: Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. ( A ) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. ( B ) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.

    Techniques Used: Variant Assay, Agarose Gel Electrophoresis, Recombinant, Staining

    2) Product Images from "Rescuing ocular development in an anophthalmic pig by blastocyst complementation"

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201808861

    Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.
    Figure Legend Snippet: Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.

    Techniques Used: In Vivo, Derivative Assay, Polymerase Chain Reaction, Negative Control, Expressing, Agarose Gel Electrophoresis

    The chimeric contributions in vitro were detected by immunofluorescence and RFLP analysis simultaneously Representative immunofluorescence images show the expression of RFP and GFP simultaneously in chimeric embryos. GFP‐labeled and RFP‐labeled blastocysts are shown as negative controls. Scale bar, 50 μm. Representative immunofluorescence images showed the expression of GFP in the chimeric blastocyst. Scale bar, 50 μm. Genotyping for porcine blastocysts derived from injected embryos. Porcine MITF ‐specific primers were used for the detection of chimeric contribution. Restriction enzymes DraI was used for digestion of PCR products. NC, negative control with no genomic DNA loaded. Mut, Bama MITF L247S/L247S blastocyst DNA loaded. WT, Bama wild‐type blastocyst DNA loaded. B1‐B10, collected the injected blastocysts DNA loaded.
    Figure Legend Snippet: The chimeric contributions in vitro were detected by immunofluorescence and RFLP analysis simultaneously Representative immunofluorescence images show the expression of RFP and GFP simultaneously in chimeric embryos. GFP‐labeled and RFP‐labeled blastocysts are shown as negative controls. Scale bar, 50 μm. Representative immunofluorescence images showed the expression of GFP in the chimeric blastocyst. Scale bar, 50 μm. Genotyping for porcine blastocysts derived from injected embryos. Porcine MITF ‐specific primers were used for the detection of chimeric contribution. Restriction enzymes DraI was used for digestion of PCR products. NC, negative control with no genomic DNA loaded. Mut, Bama MITF L247S/L247S blastocyst DNA loaded. WT, Bama wild‐type blastocyst DNA loaded. B1‐B10, collected the injected blastocysts DNA loaded.

    Techniques Used: In Vitro, Immunofluorescence, Expressing, Labeling, Derivative Assay, Injection, Polymerase Chain Reaction, Negative Control

    3) Product Images from "Conformation-Selective Methylation of Geminivirus DNA ▿"

    Article Title: Conformation-Selective Methylation of Geminivirus DNA ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05567-11

    RCA/RFLP analysis of bisulfite-treated DNA. The genome map of TYLCSV shows DraI sites which are present in the source DNA (underlined) or which may be created on the viral (v) or complementary (c) strand after bisulfite treatment. Total nucleic acids
    Figure Legend Snippet: RCA/RFLP analysis of bisulfite-treated DNA. The genome map of TYLCSV shows DraI sites which are present in the source DNA (underlined) or which may be created on the viral (v) or complementary (c) strand after bisulfite treatment. Total nucleic acids

    Techniques Used:

    Overall cytosine methylation of the TYLCSV viral strand and AbMV DNA B complementary strand. (a) Distributions of fragments obtained after sequencing of DraI-digested randomly cloned RCA products. Positions of the ORFs are marked with arrows. (b) Frequencies
    Figure Legend Snippet: Overall cytosine methylation of the TYLCSV viral strand and AbMV DNA B complementary strand. (a) Distributions of fragments obtained after sequencing of DraI-digested randomly cloned RCA products. Positions of the ORFs are marked with arrows. (b) Frequencies

    Techniques Used: Methylation, Sequencing, Clone Assay

    4) Product Images from "Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells"

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

    Journal: Biochimica et Biophysica Acta

    doi: 10.1016/j.bbamcr.2011.08.008

    Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B . (b–e) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (− S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels b–e were run in the same second dimension gel and panels b–e were produced from an X-ray film. Panels c′ and e′ are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1] )/(y/1n [− S1] ) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM. Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B. (b–e) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (− S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels b–e were run in the same second dimension gel and panels b–e were produced from an X-ray film. Panels c′ and e′ are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1] )/(y/1n [− S1] ) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM.
    Figure Legend Snippet: Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B . (b–e) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (− S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels b–e were run in the same second dimension gel and panels b–e were produced from an X-ray film. Panels c′ and e′ are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1] )/(y/1n [− S1] ) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM. Sensitivity of mtDNA replication intermediates to S1 nuclease upon DNA ligase III depletion. (A) Analysis of a DraI-digested fragment of mtDNA with two-dimensional agarose gel electrophoresis. (a) A schematic drawing of the fragment visualised by Southern hybridisation. The non-replicating molecules (1N spot) and replication intermediates (Y arc) are indicated. The molecule structure of the DraI-digested mtDNA replication intermediates at 3 different positions of Y arc is drawn in the insets with a black bar as non-replicating portion and grey bars as replicated portions of the fragment. The apex region of the Y arc used for the quantification is indicated as a grey rectangle and the region of 1N spot used for the quantification as a grey dotted circle. More information on the fragment is provided in Supplementary Fig. 1B. (b–e) The DraI-digested mtDNA fragment of scramble (Sc) dsRNA or DNA ligase III-specific (LIII) dsRNA-treated cells without (− S1) (b and c) and with S1 nuclease treatment (+ S1) (d and e). The samples in panels b–e were run in the same second dimension gel and panels b–e were produced from an X-ray film. Panels c′ and e′ are a longer exposure version of panels c and e. (B) Numerical presentation of the Y arc stability against S1 nuclease. The value of (y/1n [+ S1] )/(y/1n [− S1] ) from Sc dsRNA-treated samples is expressed as 100 and the relative value from LIII dsRNA-treated samples was calculated. Data represent the mean of 3 independent transfection experiments ± SEM.

    Techniques Used: Agarose Gel Electrophoresis, Hybridization, Produced, Transfection

    5) Product Images from "Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome"

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    Journal: Nucleic Acids Research

    doi:

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.
    Figure Legend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Techniques Used: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation

    6) Product Images from "Molecular Characterization of vanB Elements in Naturally Occurring Gut Anaerobes"

    Article Title: Molecular Characterization of vanB Elements in Naturally Occurring Gut Anaerobes

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.49.5.1688-1694.2005

    (A) Schematic diagram of the Tn 1549-van PCR product. RcaI (R) and DraI (D) sites were deduced from the sequence of Tn 1549
    Figure Legend Snippet: (A) Schematic diagram of the Tn 1549-van PCR product. RcaI (R) and DraI (D) sites were deduced from the sequence of Tn 1549

    Techniques Used: Polymerase Chain Reaction, Sequencing

    7) Product Images from "HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner"

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000669

    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Figure Legend Snippet: Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Techniques Used: Produced, DNA Synthesis, Transmission Electron Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Synthesized

    8) Product Images from "A molecular inversion probe assay for detecting alternative splicing"

    Article Title: A molecular inversion probe assay for detecting alternative splicing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-712

    A schematic of the asMIP methodology . (A) The asMIP design. Unreacted probes terminate with 3'- and 5'-interrogation sequences (no. 1) abutted by two DraI cleavage sites (no. 2). Each probe contains two unique sequence tags (no. 3) and two common primer binding sites (no. 4). (B) The asMIP assay. Step 1, sample preparation: immobilized cDNA was reverse transcribed (arrow) from polyadenylated RNA (curved lines followed by AAAAAA) using oligo-dT primers (TTTTT), which were covalently attached to magnetic beads (gray circles). Following cDNA synthesis, RNA is digested and washed away. Step 2, asMIP hybridization: unreacted asMIP probes (flattened nicked circles) terminate with 3'- and 5'-interrogation sequences (gray and colored lines), which are homologous to the exon sequences that flank splice junctions on the cDNA. Probes quantitatively anneal to the appropriate exon-exon junction (colored probes). Probes that do not hybridize (gray probes) are washed away. Step 3, asMIP circularization: bound asMIPs are ligated into circles (small arrows). Step 4, asMIP amplification and quantitation: only the successfully ligated probes (contiguous circles) can be exponentially amplified using the common PCR primers (thin black lines). Each probe contains two unique sequence tags, which are amplified by PCR for multiplexed detection via array hybridization or high-throughput sequencing (bar graph).
    Figure Legend Snippet: A schematic of the asMIP methodology . (A) The asMIP design. Unreacted probes terminate with 3'- and 5'-interrogation sequences (no. 1) abutted by two DraI cleavage sites (no. 2). Each probe contains two unique sequence tags (no. 3) and two common primer binding sites (no. 4). (B) The asMIP assay. Step 1, sample preparation: immobilized cDNA was reverse transcribed (arrow) from polyadenylated RNA (curved lines followed by AAAAAA) using oligo-dT primers (TTTTT), which were covalently attached to magnetic beads (gray circles). Following cDNA synthesis, RNA is digested and washed away. Step 2, asMIP hybridization: unreacted asMIP probes (flattened nicked circles) terminate with 3'- and 5'-interrogation sequences (gray and colored lines), which are homologous to the exon sequences that flank splice junctions on the cDNA. Probes quantitatively anneal to the appropriate exon-exon junction (colored probes). Probes that do not hybridize (gray probes) are washed away. Step 3, asMIP circularization: bound asMIPs are ligated into circles (small arrows). Step 4, asMIP amplification and quantitation: only the successfully ligated probes (contiguous circles) can be exponentially amplified using the common PCR primers (thin black lines). Each probe contains two unique sequence tags, which are amplified by PCR for multiplexed detection via array hybridization or high-throughput sequencing (bar graph).

    Techniques Used: Sequencing, Binding Assay, Sample Prep, Magnetic Beads, Hybridization, Amplification, Quantitation Assay, Polymerase Chain Reaction, Next-Generation Sequencing

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026594

    PAS77 mapping and pharynx markers. (A) Probable location of the PAS77 pharynx phenotype allele is between −4.47 cM and −3.1 cM relative to the genetic center of LG.III (red circle) derived by mapping with DraI specific SNPs corresponding to DNA clones Y71H2B, F45H7, and F56C9 (orange lines) and complementation with deficiency strains with overlapping chromosomal deletions (blue lines). (B) glp-1 RNAi resulted in an Aph phenotype in > 50% of larvae; serving as a control for RNAi effectiveness. Arrow shows region missing anterior pharynx cells. (C and D) C35D10.5 RNAi or M88.2 RNAi results in a short pharynx phenotype. (E) xbp-1 (R74.3) RNAi eliminates GFP expression in pharynx. (F) wild-type MH27 AJM-1 adherens junction antibody staining shows four distinct regions in the pharynx (arrows). (G) PAS77 MH27 antibody staining shows four compressed pharynx regions (arrows). (H) Wild-type MH4 Intermediate Filament antibody staining showing three sets of marginal cells (arrows). (I) PAS77 MH4 antibody staining showing less distinct marginal cell boundaries (arrows). Bar is ∼10 µM.
    Figure Legend Snippet: PAS77 mapping and pharynx markers. (A) Probable location of the PAS77 pharynx phenotype allele is between −4.47 cM and −3.1 cM relative to the genetic center of LG.III (red circle) derived by mapping with DraI specific SNPs corresponding to DNA clones Y71H2B, F45H7, and F56C9 (orange lines) and complementation with deficiency strains with overlapping chromosomal deletions (blue lines). (B) glp-1 RNAi resulted in an Aph phenotype in > 50% of larvae; serving as a control for RNAi effectiveness. Arrow shows region missing anterior pharynx cells. (C and D) C35D10.5 RNAi or M88.2 RNAi results in a short pharynx phenotype. (E) xbp-1 (R74.3) RNAi eliminates GFP expression in pharynx. (F) wild-type MH27 AJM-1 adherens junction antibody staining shows four distinct regions in the pharynx (arrows). (G) PAS77 MH27 antibody staining shows four compressed pharynx regions (arrows). (H) Wild-type MH4 Intermediate Filament antibody staining showing three sets of marginal cells (arrows). (I) PAS77 MH4 antibody staining showing less distinct marginal cell boundaries (arrows). Bar is ∼10 µM.

    Techniques Used: Derivative Assay, Clone Assay, Expressing, Staining

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

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

    10) Product Images from "Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs"

    Article Title: Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs

    Journal: Scientific Reports

    doi: 10.1038/srep13348

    Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.
    Figure Legend Snippet: Rapidly selecting most effective sgRNAs by single blastocyst genotyping. ( A ) Overview and timeline of the experiment. Beginning wit h experimental design, assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days. ( B ) Genotyping of single blastocyst derived from Cas9 mRNA and sgRNA-injected parthenogenetic oocytes by RFLP and Sanger sequence analyses. Representative RFLP agarose gel electrophoresis showing PCR product of target region derived from 10 individual blastocysts digested with different restriction enzymes. The F1, F2, R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI, HpyCH4V, BsaJI and DraI, respectively. In each RFLP assay, some PCR products were sequenced to confirm the mutations at the target sites. The numbers on the right show the type of mutation and how many nucleotides are involved, with “−” and “+” indicating deletion or insertion of the given number of nucleotides, respectively. The sgRNA sequence is labeled in red, and the PAM sequence is labeled in purple. Deleted bases are marked with colons, and arrows indicate the sites of inserted bases, which are listed under the mutant alleles.

    Techniques Used: Mutagenesis, Derivative Assay, Injection, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, RFLP Assay, Labeling

    Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.
    Figure Legend Snippet: Genotyping of single fibroblast colonies derived from FACS sorting based on the expression of EGFP fluorescence by RFLP analysis. Agarose gel electrophoresis showing PCR product of target region digested with different restriction enzymes. The R1 and R2 sgRNA mutagenesis efficiencies were assessed by BsaJI and DraI, respectively.

    Techniques Used: Derivative Assay, FACS, Expressing, Fluorescence, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Mutagenesis

    11) Product Images from "Rescuing ocular development in an anophthalmic pig by blastocyst complementation"

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201808861

    Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs A Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. B DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.
    Figure Legend Snippet: Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs A Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. B DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.

    Techniques Used: In Vivo, Derivative Assay, Polymerase Chain Reaction, Negative Control, Expressing, Agarose Gel Electrophoresis

    12) Product Images from "Context Dependence of Trinucleotide Repeat Structures"

    Article Title: Context Dependence of Trinucleotide Repeat Structures

    Journal: Biochemistry

    doi: 10.1021/bi902043u

    Image of a nondenaturing 12% polyacrylamide gel to assess restriction digestion of three-way junctions. Lanes 1 and 8 contain a 5-base pair ladder with 15 and 50 base pair oligonucleotides marked with arrows. Lanes 2-4 correspond to the unmodified three-way junction without 2-aminopurine. Lanes 2 and 3 represent the products of digestion by DraI and BsaAI, respectively, and the mobilities of the faster bands are between the 15 and 20 base pairs. Lane 4 shows the three-way junction before digestion. Lanes 5-7 correspond to the modified three-way junction 3-CAG, and the mobilities of the faster bands are between the 15 and 20 base pairs. Lanes 5 and 6 represent the products of digestion by DraI and BsaAI, respectively. Lane 7 shows the three-way junction before digestion.
    Figure Legend Snippet: Image of a nondenaturing 12% polyacrylamide gel to assess restriction digestion of three-way junctions. Lanes 1 and 8 contain a 5-base pair ladder with 15 and 50 base pair oligonucleotides marked with arrows. Lanes 2-4 correspond to the unmodified three-way junction without 2-aminopurine. Lanes 2 and 3 represent the products of digestion by DraI and BsaAI, respectively, and the mobilities of the faster bands are between the 15 and 20 base pairs. Lane 4 shows the three-way junction before digestion. Lanes 5-7 correspond to the modified three-way junction 3-CAG, and the mobilities of the faster bands are between the 15 and 20 base pairs. Lanes 5 and 6 represent the products of digestion by DraI and BsaAI, respectively. Lane 7 shows the three-way junction before digestion.

    Techniques Used: Modification

    13) Product Images from "Functional epialleles at an endogenous human centromere"

    Article Title: Functional epialleles at an endogenous human centromere

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1203126109

    CENP-A location on HSA17 and D17Z1 HOR polymorphisms. ( A ) The D17Z1 HOR contains 16 monomeric repeats (16-mer) and is defined by EcoRI restriction sites (E). A 3-monomer deletion generates a 13-mer from the 16-mer. Some HORs also contain sequence polymorphisms that create DraI (D) and additional EcoRI restriction sites. Part of the HOR (gray arrowheads) was amplified by PCR and digested with DraI. If the DraI site is present, the 850-bp PCR product is reduced to 750-bp. Each HOR variant can be correlated with the 850-bp (16-mer) or 750-bp (13-mer) bands, and the intensity of bands indicates relative amounts of each repeat form. ( B ) In the CEPH1345 family, 16-mer and 13-mer D17Z1 variants were present in each individual. Functional homozygotes (Z1/Z1, with CENP-A located at D17Z1 on both HSA17s), such as individual 13 , had darker upper bands, indicating a greater proportion of 16-mers. Functional heterozygotes (Z1/Z1-B), such as individual 07, typically had darker lower bands or equivalently dark upper and lower bands. NT, not tested. Because CEPH lines were diploid, proportions of each variant could not be assigned to specific HSA17s. Thus, single HSA17s in somatic cell hybrids were genotyped. ( C ) In SCHL-13A, SCHL-14A, and SCHL1-H1 that have CENP-A at D17Z1, only the 16-mer was detected. In SCHL1-H2, which shows CENP-A at D17Z1-B, the 13-mer and 16-mer were equally present.
    Figure Legend Snippet: CENP-A location on HSA17 and D17Z1 HOR polymorphisms. ( A ) The D17Z1 HOR contains 16 monomeric repeats (16-mer) and is defined by EcoRI restriction sites (E). A 3-monomer deletion generates a 13-mer from the 16-mer. Some HORs also contain sequence polymorphisms that create DraI (D) and additional EcoRI restriction sites. Part of the HOR (gray arrowheads) was amplified by PCR and digested with DraI. If the DraI site is present, the 850-bp PCR product is reduced to 750-bp. Each HOR variant can be correlated with the 850-bp (16-mer) or 750-bp (13-mer) bands, and the intensity of bands indicates relative amounts of each repeat form. ( B ) In the CEPH1345 family, 16-mer and 13-mer D17Z1 variants were present in each individual. Functional homozygotes (Z1/Z1, with CENP-A located at D17Z1 on both HSA17s), such as individual 13 , had darker upper bands, indicating a greater proportion of 16-mers. Functional heterozygotes (Z1/Z1-B), such as individual 07, typically had darker lower bands or equivalently dark upper and lower bands. NT, not tested. Because CEPH lines were diploid, proportions of each variant could not be assigned to specific HSA17s. Thus, single HSA17s in somatic cell hybrids were genotyped. ( C ) In SCHL-13A, SCHL-14A, and SCHL1-H1 that have CENP-A at D17Z1, only the 16-mer was detected. In SCHL1-H2, which shows CENP-A at D17Z1-B, the 13-mer and 16-mer were equally present.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction, Variant Assay, Functional Assay

    14) Product Images from "Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene"

    Article Title: Generation and Characterization of a Transgenic Pig Carrying a DsRed-Monomer Reporter Gene

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0106864

    Transgenic founder pigs carrying the DsRed gene were detected by (a) PCR and (b) Southern blot analysis. The presence of transgenes in DsRed2 pigs were confirmed by using a DsRed primer, which produced a 780-bp PCR product. The genomic DNA of these 2 transgenic pigs (Nos. 1 and 3) were digested using XbaI and DraI. The digested DNA was subsequently hybridized using a 1.1-kb probe.
    Figure Legend Snippet: Transgenic founder pigs carrying the DsRed gene were detected by (a) PCR and (b) Southern blot analysis. The presence of transgenes in DsRed2 pigs were confirmed by using a DsRed primer, which produced a 780-bp PCR product. The genomic DNA of these 2 transgenic pigs (Nos. 1 and 3) were digested using XbaI and DraI. The digested DNA was subsequently hybridized using a 1.1-kb probe.

    Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Southern Blot, Produced

    The DsRed-Monomer transgenic construct pCX-DsRed-Monomer. Arrows indicate the positions of the PCR DsRed primers. XbaI and DraI were restriction enzyme digestion sites. The thick black line indicates the position of the Southern blot probe.
    Figure Legend Snippet: The DsRed-Monomer transgenic construct pCX-DsRed-Monomer. Arrows indicate the positions of the PCR DsRed primers. XbaI and DraI were restriction enzyme digestion sites. The thick black line indicates the position of the Southern blot probe.

    Techniques Used: Transgenic Assay, Construct, Polymerase Chain Reaction, Southern Blot

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Circular RNA biogenesis can proceed through an exon-containing lariat precursor
    Article Snippet: .. 20 U of Dra I (NEB) was added directly to the 50 μl PCR reaction, and the reaction was allowed to incubate at 37°C for 1 hr. .. From there, the PCR was resumed for 35 additional cycles without purification. qPCR reactions were assembled as 10 μl reactions using AccuPower 2X GreenStar qPCR Master Mix (Bioneer) and Power SYBR Green Master Mix (Life Technologies) with 0.3 μl of template used per reaction.

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation
    Article Snippet: DNA samples were analyzed using PCR with specific primers for the MITF gene and restriction fragment length polymorphism (RFLP) analysis. .. Eight microliters of PCR product was digested with DraI (New England Biolabs, USA). .. The full‐length reaction products were 471 bp and were separated into 261 and 210 bp by 2% agarose gel electrophoresis in the presence of ethidium bromide solution and visualized with a UV transilluminator (UVP, Upland, CA, USA).

    Agarose Gel Electrophoresis:

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Southern Blot:

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation
    Article Snippet: Generation of T2/Onc2.3 transgenic mice pT2/Onc2.3 DNA was linearized with restriction enzyme with Sca I (NEB #R3122) at position 3,502 bp ( ) at 37°C for 90 m, followed by heat inactivation at 80 °C for 20 m. pT2/Onc2.3:Sca I linear plasmid, at 2 ng/uL, 4 ng/uL and 10 ng/uL, was prepared for microinjection into (B6C3)F2 hybrid embryos using standard techniques ( ). .. Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods. .. Individual transgenic lines (n=5) were established by backcrossing founders to C57BL/6J (JAX#000664).

    Incubation:

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1
    Article Snippet: Briefly, the heteroduplex phagemid DNA substrate (48 fmol) containing a T/G mismatch in its unique SalI site and a single nick generated by Nt·BstNBI 361 nucleotides 5′ from the mispaired T was incubated with 100 μg of nuclear extracts of HEK293 cells pretreated or not with EXO1 siRNA and supplemented with 40 nM Exo1 wt, E109K or D173A in 20 mM Tris·HCl pH 7.6, 110 mM KCl, 5 mM MgCl2 , 1 mM glutathione, 1.5 mM ATP, 50 μg/ml BSA and 100 μM dNTPs for 30 min in a total volume of 25 μl. .. The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB). .. RNase A (40 ng, Sigma-Aldrich) was then added and, following an overnight incubation at 37°C, the reaction products were separated on a 1% agarose gel eluted with TAE buffer and stained with GelRed.

    Article Title: Analysis of Distant Communication on Defined Chromatin Templates In Vitro
    Article Snippet: However a similar technique could be applied to any DNA region on the plasmid ( , ). .. 750 ng of DNA or nucleosomal templates (20 μg/ml) were incubated in the presence of an excess of Dra I and Bgl II restriction endonucleases (10 units each) in the NEBuffer #2 at 37°C for 2 hours. .. DNA was purified by phenol/chloroform extraction followed by ethanol precipitation and analyzed by electrophoresis in 1% agarose/TAE gel.

    Modification:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Quantification of mtDNA was conducted using a rt-qPCR method essentially as described . .. 2.4 DNA modification, 2D-AGE and Southern hybridisation Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2). .. In some cases the digested DNA was further modified with 9.5 unit S1 nuclease (Promega) in 30 μl reaction mixture at 37 °C for 20 min and then 1.5 μl of 0.5 M EDTA (pH 8) was added to the reaction.

    Hybridization:

    Article Title: Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells
    Article Snippet: Quantification of mtDNA was conducted using a rt-qPCR method essentially as described . .. 2.4 DNA modification, 2D-AGE and Southern hybridisation Total DNA was digested with DraI (New England Biolabs), and then precipitated and suspended in 10 mM Hepes-NaOH (pH 7.2). .. In some cases the digested DNA was further modified with 9.5 unit S1 nuclease (Promega) in 30 μl reaction mixture at 37 °C for 20 min and then 1.5 μl of 0.5 M EDTA (pH 8) was added to the reaction.

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    New England Biolabs dra i
    Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic <t>DNA</t> digested to completion with <t>Dra</t> I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.
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    Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.

    Journal: bioRxiv

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

    doi: 10.1101/2020.08.17.254565

    Figure Lengend Snippet: Transgenic SB-T2/Onc2.3 founder mice. ( a ) Plasmid map of pT2/Onc2.3-MBM102, denoting essential features and base pair position in parenthesis, used to create the T2/Onc2.3 high-copy transgenic lines. ( b ) Southern blot of T2/Onc2.3 founder mice using a probe corresponding to the En2 -SA ( Engrailed 2 splice acceptor) element within the SB-T2/SB-GT allele construct that also cross hybridizes to the endogenous En2 mouse gene on chromosome 5. Genomic DNA digested to completion with Dra I restriction enzyme and detected with a radioisotope- 32 P labeled En2 -SA probe identifies the diploid endogenous En2 locus as a discrete 2.1 kb band and any all copies of the T2/SB-GT monomer that have been liberated from the their randomly inserted transgenic loci as a discrete 1.0 kb band. Mice with multi-copy concatemer alleles are identified as containing darker staining bands relative to the two copies of the En2 endogenous locus band. Blue arrows and dotted boxes denote animals selected for germ line breeding — five separate transgenic founders, three females (TG.14885, TG.14922, and TG.14942) and two males (TG.14913 and TG.14927), were selected for mating with wildtype C57BL/6J mice to confirm the germ line transmission of the SB-T/2SB-GT concatemer alleles to progeny. Mice denoted with blue dotted boxes produced transgenic carrier pups with a transgene copy numbers that differed between individuals, suggesting the presence of two or more independent transgenic concatemer integration sites. Mice denoted with red dotted boxes produced transgenic carrier pups with a transgene copy number that did not differ between individuals, suggesting that they resulted from a single transgenic concatemer integration site. Lines established from founders TG.14913, TG.14922, and TG.14942 were expanded and bred for further experiments. Bam HI restriction digested lambda (ƛ)-phage DNA is provided as a reference, a small amount was added to the radioisotope- 32 P labeling reaction with the En2 -SA probe to show detection on the developed Southern blot.

    Article Snippet: Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods.

    Techniques: Transgenic Assay, Mouse Assay, Plasmid Preparation, Southern Blot, Construct, Labeling, Staining, Transmission Assay, Produced

    Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.

    Journal: bioRxiv

    Article Title: Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

    doi: 10.1101/2020.08.17.254565

    Figure Lengend Snippet: Estimating SB transposon concatemer copy number from different SB strains. ( a ) Inverse image of EtBr stained agarose gel (1.2% TBE, run at 30 volts for 30 hours) containing genomic DNA digested with Dra I for 16 hours at 37 °C for Southern blot analysis. Molecular weight markers: left, lambda cut with Hin DIII; right, 100 bp ladder. ( b ) Southern blot demonstrating relative concatemer copy number differences between high-copy and low-copy SB transposon alleles in homozygous mice with unmobilized transposons. The En2 site provides a reference for a single copy, diploid gene.

    Article Snippet: Tail biopsy genomic DNA from founder animals was digested with Dra I (NEB) and Bam HI (NEB), run on a 0.8% TAE agarose gel at 30 v for 16 h, transferred to membrane, and screened by Southern blotting using a 32 P-labeled En2-SA (splice acceptor) probe, a 500 bp En2-SA PCR product with pT2/Onc2.3 template and primers T2.3-En2.5Probe, 5’-GCTGCAATAAACAAGTTGGCCG-3’ and T2.3-En2.3Probe, 5’-CTTGGGTCAAACATTTCGAGTAGCC-3’, and standard methods.

    Techniques: Staining, Agarose Gel Electrophoresis, Southern Blot, Molecular Weight, Mouse Assay

    Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. ( A ) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. ( B ) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Biochemical characterization of a cancer-associated E109K missense variant of human exonuclease 1

    doi: 10.1093/nar/gku419

    Figure Lengend Snippet: Wild-type EXO1 and its E109K variant complement the MMR defect of EXO1-depleted extracts of HEK293 cells. ( A ) Schematic representation of the T/G MMR substrate. The SalI restriction site contains a T/G mismatch, which renders the site refractory to cleavage. Repair of the T/G mismatch to C/G restores a bona fide SalI site. The SalI- and the three DraI restriction sites are indicated. The restriction patterns seen upon agarose gel electrophoresis before and after repair are shown on the right. The Nt.BstNBI nicking site is located 316 nucleotides 5′ from the mispaired T. ( B ) Mismatch repair assay using HEK293 siEXO1 extracts supplemented with recombinant EXO1, either wild-type, or the E109K or D173A variants. The reactions were stopped after 30 min and the recovered substrates were digested with SalI/DraI. In the absence of repair, the substrate gives rise to fragments of 2484, 694 and 19 bp, while the repaired substrate generates fragments of 1324, 1160, 694 and 19 bp. The figure shows a scan of a 1% agarose gel stained with GelRed. The image is representative of three independent experiments.

    Article Snippet: The reactions were terminated by a 30-min incubation with a stop solution (final concentrations: 0.5 mM EDTA, 1.5% SDS (sodium dodecyl sulfate), 2.5 mg/ml proteinase K), cleaned up on a MinElute column (Qiagen), and the recovered phagemid was subjected to restriction digest with 6 U SalI and 20 U DraI (NEB).

    Techniques: Variant Assay, Agarose Gel Electrophoresis, Recombinant, Staining

    Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Analysis of Distant Communication on Defined Chromatin Templates In Vitro

    doi: 10.1007/978-1-60327-015-1_33

    Figure Lengend Snippet: Characterization of chromatin templates using restriction enzyme sensitivity assay. Chromatin was assembled on supercoiled pYP05 plasmid and digested with an excess of restriction enzymes Dra I and Bgl II. Then DNA was purified and analyzed in 1% agarose

    Article Snippet: 750 ng of DNA or nucleosomal templates (20 μg/ml) were incubated in the presence of an excess of Dra I and Bgl II restriction endonucleases (10 units each) in the NEBuffer #2 at 37°C for 2 hours.

    Techniques: Sensitive Assay, Plasmid Preparation, Purification

    Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation

    doi: 10.15252/emmm.201808861

    Figure Lengend Snippet: Generation of E44 chimeric porcine fetus in vivo by complementation of MITF L247S/L247S embryos with donor blastomeres derived from LW PEFs Four fetuses (named NW‐1, NW‐2, NW‐3, and NW‐4) with different eye morphology were retrieved at E44 by Caesarean section. The arrows identify the melanin in the eye. DraI digestion of PCR products from the multiple organs of the four fetuses (top). NC, negative control with no genomic DNA loaded. Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Multiple organs of the four fetuses were examined for KIT expression by agarose gel electrophoresis (bottom). Mut, MITF L247S/L247S genomic DNA loaded. WT, LW genomic DNA loaded. Source data are available online for this figure.

    Article Snippet: Eight microliters of PCR product was digested with DraI (New England Biolabs, USA).

    Techniques: In Vivo, Derivative Assay, Polymerase Chain Reaction, Negative Control, Expressing, Agarose Gel Electrophoresis

    The chimeric contributions in vitro were detected by immunofluorescence and RFLP analysis simultaneously Representative immunofluorescence images show the expression of RFP and GFP simultaneously in chimeric embryos. GFP‐labeled and RFP‐labeled blastocysts are shown as negative controls. Scale bar, 50 μm. Representative immunofluorescence images showed the expression of GFP in the chimeric blastocyst. Scale bar, 50 μm. Genotyping for porcine blastocysts derived from injected embryos. Porcine MITF ‐specific primers were used for the detection of chimeric contribution. Restriction enzymes DraI was used for digestion of PCR products. NC, negative control with no genomic DNA loaded. Mut, Bama MITF L247S/L247S blastocyst DNA loaded. WT, Bama wild‐type blastocyst DNA loaded. B1‐B10, collected the injected blastocysts DNA loaded.

    Journal: EMBO Molecular Medicine

    Article Title: Rescuing ocular development in an anophthalmic pig by blastocyst complementation

    doi: 10.15252/emmm.201808861

    Figure Lengend Snippet: The chimeric contributions in vitro were detected by immunofluorescence and RFLP analysis simultaneously Representative immunofluorescence images show the expression of RFP and GFP simultaneously in chimeric embryos. GFP‐labeled and RFP‐labeled blastocysts are shown as negative controls. Scale bar, 50 μm. Representative immunofluorescence images showed the expression of GFP in the chimeric blastocyst. Scale bar, 50 μm. Genotyping for porcine blastocysts derived from injected embryos. Porcine MITF ‐specific primers were used for the detection of chimeric contribution. Restriction enzymes DraI was used for digestion of PCR products. NC, negative control with no genomic DNA loaded. Mut, Bama MITF L247S/L247S blastocyst DNA loaded. WT, Bama wild‐type blastocyst DNA loaded. B1‐B10, collected the injected blastocysts DNA loaded.

    Article Snippet: Eight microliters of PCR product was digested with DraI (New England Biolabs, USA).

    Techniques: In Vitro, Immunofluorescence, Expressing, Labeling, Derivative Assay, Injection, Polymerase Chain Reaction, Negative Control