exonuclease thermoscientific digestion  (Thermo Fisher)


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    Name:
    Exonuclease I 20 U µL
    Description:
    Thermo Scientific Exonuclease I ExoI degrades single stranded DNA in a 3 →5 direction It releases deoxyribonucleoside 5 monophosphates in a stepwise manner and leaves 5 terminal dinucleotides intact It does not cleave DNA strands with terminal 3 OH groups blocked by phosphoryl or acetyl groups Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures • prior to PCR product sequencing• for one tube megaprimer PCR mutagenesis• Removal of single stranded DNA containing a 3 hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single stranded DNA with a 3 hydroxyl terminusNoteThe enzyme is not suitable for removing 3 overhangs of dsDNA
    Catalog Number:
    EN0581
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|PCR Cloning|Mutagenesis
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    Structured Review

    Thermo Fisher exonuclease thermoscientific digestion
    Thermo Scientific Exonuclease I ExoI degrades single stranded DNA in a 3 →5 direction It releases deoxyribonucleoside 5 monophosphates in a stepwise manner and leaves 5 terminal dinucleotides intact It does not cleave DNA strands with terminal 3 OH groups blocked by phosphoryl or acetyl groups Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures • prior to PCR product sequencing• for one tube megaprimer PCR mutagenesis• Removal of single stranded DNA containing a 3 hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single stranded DNA with a 3 hydroxyl terminusNoteThe enzyme is not suitable for removing 3 overhangs of dsDNA
    https://www.bioz.com/result/exonuclease thermoscientific digestion/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease thermoscientific digestion - by Bioz Stars, 2021-07
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    Related Articles

    Fluorescence:

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae
    Article Snippet: .. Quantitative determination of SXT-Exo double strand DNA exonuclease activities using the sensitive fluorescent PicoGreen reagent We quantified the dsDNA exonuclease activities of SXT-Exo under a variety of different experimental conditions using sensitive fluorescence assays incorporating the PicoGreen reagent (Invitrogen). ..

    Plasmid Preparation:

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates
    Article Snippet: Hybridization signals were detected after exposure to an image plate and read-out by phosphorimaging (FLA-3000, Fujifilm). .. Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific). ..

    Article Title: Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense
    Article Snippet: .. The E. coli Exo I gene was amplified from the genomic DNA of E. coli strain BL21 (DE3) and ligated into pET21a overexpression vector (Invitrogen) with a 6*his-tag appended on the C-terminus of the protein. .. Protein expression and purification The E. coli strains harboring the MszExo I (or E. coli Exo I) expression plasmid were grown at 37°C in LB medium containing 100 μg/ml ampicillin.

    DNA Extraction:

    Article Title: The HIV-1 provirus excised by a single CRISPR/Cas9 RNA guide persists in the host cell and may be reactivated
    Article Snippet: Selected clones were then transfected with T5 gRNApspCas9 plasmid (Cat. No. 459 Addgene) and cultivated in the presence of Puromycin (3 μg/ml) to select for the transfected cells. .. Following DNA extraction and digestion with ATP-dependent exonuclease, 50-200 ng of total DNA, quantitated before exonuclease digestion, were used to transform 108 ultracompetent Stbl2 E. coli cells (Invitrogen, Carlsbad, CA, USA). .. Transformants were seeded in Luria Bertani (LB) agar plates supplemented with Kanamycin (50 μg/ml).

    Amplification:

    Article Title: Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense
    Article Snippet: .. The E. coli Exo I gene was amplified from the genomic DNA of E. coli strain BL21 (DE3) and ligated into pET21a overexpression vector (Invitrogen) with a 6*his-tag appended on the C-terminus of the protein. .. Protein expression and purification The E. coli strains harboring the MszExo I (or E. coli Exo I) expression plasmid were grown at 37°C in LB medium containing 100 μg/ml ampicillin.

    Over Expression:

    Article Title: Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense
    Article Snippet: .. The E. coli Exo I gene was amplified from the genomic DNA of E. coli strain BL21 (DE3) and ligated into pET21a overexpression vector (Invitrogen) with a 6*his-tag appended on the C-terminus of the protein. .. Protein expression and purification The E. coli strains harboring the MszExo I (or E. coli Exo I) expression plasmid were grown at 37°C in LB medium containing 100 μg/ml ampicillin.

    Ethanol Precipitation:

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects
    Article Snippet: .. Exonuclease I treatment was performed overnight at 37°C with 200 U exonuclease I (Thermo Fisher Scientific) per 5 µg DNA in 500 µl, followed by ethanol precipitation. ..

    Southern Blot:

    Article Title: Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes
    Article Snippet: PS6 is a 0.4-kb PCR fragment amplified from TCRαsJ/sJ genomic DNA using the PS6-1 and PS6-2 oligonucleotides (Table S1, available at http://www.jem.org/cgi/content/full/jem.20061460/DC1 ). .. For exonuclease V digestion, 15 μg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses. .. For mung bean nuclease assays, 15 μg of thymocyte DNA was digested with 12.5 U of mung bean nuclease (New England Biolabs, Inc.) in 200 μl using the manufacturer's buffer supplemented with 0.4 mM ZnSO4 at 25°C for 1 h before treatment with exonuclease V, as described in this section.

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    Thermo Fisher atp dependent exonuclease
    The excised HIV-1 provirus persists and circularizes after CRISPR/Cas9-transfection. a, PCR analysis of β-globin gene in genomic <t>DNA</t> samples from NL4-3/Luc-transduced 293T cells extracted at day 2, 6, 10, 14 and 18 post CRISPR/Cas9 transfection, before (−) and after (+) <t>ATP-dependent</t> DNA exonuclease digestion. M: Molecular weight marker. b, PCR analysis of gag and pol sequences of genomic DNA treated as in a with ATP-dependent DNA exonuclease digestion. c, Rolling circle amplification (RCA) of DNA samples extracted from NL4-3/Luc-transduced at day 10 post-CRISPR/Cas9 and T5 or SC gRNAs transfection. This technique allows selective amplification of circularized DNA as concatemers, requiring digestion with a single cutter to obtain full length fragments. RCA amplicons were electrophoresed as such (−) or after EcoR I digestion (+), which cuts NL4-3/Luc once in pol . 293 T were used as a negative control. d, Schematic of circular molecules of HIV-1 provirus formed by a single molecule or two molecules bound together in sense-to-sense or sense-to-antisense orientation and size of the amplicons of interest written in red and generated by the primers indicated. e, PCR fragments obtained from RCA amplicons from 7 different experiments (#RCA1-7) using the primers shown in Fig. 2d . f, Alignment of LTR sequencing of the 750-bp fragments obtained from #RCA1-7 and retrieved from the agarose gel of Fig. 2e . The red sequences indicate the T5 gRNA annealing site. Dashes indicate base deletions and blue letters denote nucleotide mismatches compared to wild-type pNL4-3 sequence.
    Atp Dependent Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/atp dependent exonuclease/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    96
    Thermo Fisher λ exonuclease
    S1 nuclease protection mapping of the ABCA2 transcription start site. A 5′-end phosphorylated 479 bp double-stranded DNA template for the S1 probe was generated by PCR and digested with <t>λ</t> exonuclease to generate an antisense single-stranded DNA probe that was end labeled with [γ- 32 P]ATP and hybridized to total RNA from BE(2)-M17 neuroblastoma cells. Following digestion with S1 nuclease, the protected fragment was separated by electrophoresis in a 6% polyacrylamide–7 M urea gel followed by autoradiography. The antisense strand of the 479 bp DNA template was manually sequenced and the φX174 Hin f DNA ladder was radiolabeled with [γ- 32 P]ATP to serve as sequence and size markers respectively. An arrow indicates a major protected fragment of 152 bp, located 95 bp upstream of the ATG translation start site that represents the major transcription start site.
    λ Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/λ exonuclease/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
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    The excised HIV-1 provirus persists and circularizes after CRISPR/Cas9-transfection. a, PCR analysis of β-globin gene in genomic DNA samples from NL4-3/Luc-transduced 293T cells extracted at day 2, 6, 10, 14 and 18 post CRISPR/Cas9 transfection, before (−) and after (+) ATP-dependent DNA exonuclease digestion. M: Molecular weight marker. b, PCR analysis of gag and pol sequences of genomic DNA treated as in a with ATP-dependent DNA exonuclease digestion. c, Rolling circle amplification (RCA) of DNA samples extracted from NL4-3/Luc-transduced at day 10 post-CRISPR/Cas9 and T5 or SC gRNAs transfection. This technique allows selective amplification of circularized DNA as concatemers, requiring digestion with a single cutter to obtain full length fragments. RCA amplicons were electrophoresed as such (−) or after EcoR I digestion (+), which cuts NL4-3/Luc once in pol . 293 T were used as a negative control. d, Schematic of circular molecules of HIV-1 provirus formed by a single molecule or two molecules bound together in sense-to-sense or sense-to-antisense orientation and size of the amplicons of interest written in red and generated by the primers indicated. e, PCR fragments obtained from RCA amplicons from 7 different experiments (#RCA1-7) using the primers shown in Fig. 2d . f, Alignment of LTR sequencing of the 750-bp fragments obtained from #RCA1-7 and retrieved from the agarose gel of Fig. 2e . The red sequences indicate the T5 gRNA annealing site. Dashes indicate base deletions and blue letters denote nucleotide mismatches compared to wild-type pNL4-3 sequence.

    Journal: bioRxiv

    Article Title: The HIV-1 provirus excised by a single CRISPR/Cas9 RNA guide persists in the host cell and may be reactivated

    doi: 10.1101/2020.11.16.384180

    Figure Lengend Snippet: The excised HIV-1 provirus persists and circularizes after CRISPR/Cas9-transfection. a, PCR analysis of β-globin gene in genomic DNA samples from NL4-3/Luc-transduced 293T cells extracted at day 2, 6, 10, 14 and 18 post CRISPR/Cas9 transfection, before (−) and after (+) ATP-dependent DNA exonuclease digestion. M: Molecular weight marker. b, PCR analysis of gag and pol sequences of genomic DNA treated as in a with ATP-dependent DNA exonuclease digestion. c, Rolling circle amplification (RCA) of DNA samples extracted from NL4-3/Luc-transduced at day 10 post-CRISPR/Cas9 and T5 or SC gRNAs transfection. This technique allows selective amplification of circularized DNA as concatemers, requiring digestion with a single cutter to obtain full length fragments. RCA amplicons were electrophoresed as such (−) or after EcoR I digestion (+), which cuts NL4-3/Luc once in pol . 293 T were used as a negative control. d, Schematic of circular molecules of HIV-1 provirus formed by a single molecule or two molecules bound together in sense-to-sense or sense-to-antisense orientation and size of the amplicons of interest written in red and generated by the primers indicated. e, PCR fragments obtained from RCA amplicons from 7 different experiments (#RCA1-7) using the primers shown in Fig. 2d . f, Alignment of LTR sequencing of the 750-bp fragments obtained from #RCA1-7 and retrieved from the agarose gel of Fig. 2e . The red sequences indicate the T5 gRNA annealing site. Dashes indicate base deletions and blue letters denote nucleotide mismatches compared to wild-type pNL4-3 sequence.

    Article Snippet: Following DNA extraction and digestion with ATP-dependent exonuclease, 50-200 ng of total DNA, quantitated before exonuclease digestion, were used to transform 108 ultracompetent Stbl2 E. coli cells (Invitrogen, Carlsbad, CA, USA).

    Techniques: CRISPR, Transfection, Polymerase Chain Reaction, Molecular Weight, Marker, Amplification, Negative Control, Generated, Sequencing, Agarose Gel Electrophoresis

    HIV intermolecular concatemers can be isolated in large scale. a, Schematic of the approach devised to select and identify inter-molecular concatemers. 293T cells were transduced at 5 MOI with both HIV-Ori and HIV-KanR. A week later, their HIV-1 provirus was excised by CRISPR/Cas9 + T5 gRNA and the edited cells were selected by Puromycin. Then, their genomic DNA was extracted, digested by ATP-dependent exonuclease to eliminate linear DNA and used to transform bacteria. The recombinant bacteria were then selected on Kanamycin-containing agar plates, where only intermolecular joining brought about growing colonies. b, Graphic map of NL4-3/Luc/Ori and NL4-3/Luc/KanR as obtained by inserting the low-copy bacterial origin of replication SC101 (Ori) or Kanamycin resistance gene ( Kan R) in the pNL4-3/Luc backbone. c, Evaluation of transducing ability of lentiviral vectors as determined by luciferase activity of 293T cells transduced with VSV-G-pseudotyped NL4-3/Luc/Ori and NL4-3/Luc/KanR as compared to NL4-3/Luc, NL4-3/GFP and non-transduced 293T cells. d, Screening of 11 cell clones double-positive for NL4-3/Luc/Ori and NL4-3/Luc/KanR obtained by limiting dilution. For PCR, primers annealing to SC101 or KanR and pNL4-3 were used. e, PCR amplification of the LTR junctions from NL4-3/Luc/KanR/Ori double-positive cell clones a-e. f, Sequence analysis of a-e amplicons obtained in e . The red sequences indicate the T5 gRNA annealing site. Dashes indicate base deletions and green letters denote nucleotide mismatches compared to wild-type pNL4-3 LTR sequence. g, Schematic of inter-molecular concatemers bound in sense-sense orientation, as determined by nucleotide sequencing.

    Journal: bioRxiv

    Article Title: The HIV-1 provirus excised by a single CRISPR/Cas9 RNA guide persists in the host cell and may be reactivated

    doi: 10.1101/2020.11.16.384180

    Figure Lengend Snippet: HIV intermolecular concatemers can be isolated in large scale. a, Schematic of the approach devised to select and identify inter-molecular concatemers. 293T cells were transduced at 5 MOI with both HIV-Ori and HIV-KanR. A week later, their HIV-1 provirus was excised by CRISPR/Cas9 + T5 gRNA and the edited cells were selected by Puromycin. Then, their genomic DNA was extracted, digested by ATP-dependent exonuclease to eliminate linear DNA and used to transform bacteria. The recombinant bacteria were then selected on Kanamycin-containing agar plates, where only intermolecular joining brought about growing colonies. b, Graphic map of NL4-3/Luc/Ori and NL4-3/Luc/KanR as obtained by inserting the low-copy bacterial origin of replication SC101 (Ori) or Kanamycin resistance gene ( Kan R) in the pNL4-3/Luc backbone. c, Evaluation of transducing ability of lentiviral vectors as determined by luciferase activity of 293T cells transduced with VSV-G-pseudotyped NL4-3/Luc/Ori and NL4-3/Luc/KanR as compared to NL4-3/Luc, NL4-3/GFP and non-transduced 293T cells. d, Screening of 11 cell clones double-positive for NL4-3/Luc/Ori and NL4-3/Luc/KanR obtained by limiting dilution. For PCR, primers annealing to SC101 or KanR and pNL4-3 were used. e, PCR amplification of the LTR junctions from NL4-3/Luc/KanR/Ori double-positive cell clones a-e. f, Sequence analysis of a-e amplicons obtained in e . The red sequences indicate the T5 gRNA annealing site. Dashes indicate base deletions and green letters denote nucleotide mismatches compared to wild-type pNL4-3 LTR sequence. g, Schematic of inter-molecular concatemers bound in sense-sense orientation, as determined by nucleotide sequencing.

    Article Snippet: Following DNA extraction and digestion with ATP-dependent exonuclease, 50-200 ng of total DNA, quantitated before exonuclease digestion, were used to transform 108 ultracompetent Stbl2 E. coli cells (Invitrogen, Carlsbad, CA, USA).

    Techniques: Isolation, CRISPR, Recombinant, Luciferase, Activity Assay, Transduction, Clone Assay, Polymerase Chain Reaction, Amplification, Sequencing

    Increased TCRα sJ locus CE accumulation in Atm −/− thymocytes. (A) Southern analysis strategy showing the relative positions of the NsiI (N) and StuI (S) sites, PS6, and the CαI probe. Fragment sizes generated by germline TCRα sJ alleles and Jα56 and Jα61 CEs are indicated. The schematics are not drawn to scale. (B) Southern blot analyses of total thymocyte DNA and kidney DNA (K) digested with either NsiI or HincII and hybridized to the CαI probe or PS6. Bands corresponding to Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline fragment is indicated by an asterisk. Hybridization of StuI and NsiI digested DNA to PR2 is shown as a DNA loading control. The molecular mass markers (in kb) are indicated. (C) LMPCR analysis of Jα56 and Jα61 CEs, as described in Materials and methods. Shown are fivefold serial dilutions of linker-ligated samples. IL-2 gene PCR is also shown as a DNA loading control. (D and E) Exonuclease V sensitivity of Jα56 CEs was assayed by hybridizing the membrane from Fig. 3D to the CαI probe. Bands corresponding to the Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline TCRα sJ allele is indicated by an asterisk. The positions of the molecular mass markers (in kb) are indicated (D). Retention of the Jα56 CE band in exonuclease V–treated samples was quantified as described in Materials and methods (E). (F and G) TCRα sJ/sJ :Atm − / − thymocyte DNA was treated with mung bean nuclease followed by exonuclease V before digestion with StuI and hybridization to the CαI probe, as described in Materials and methods. Fragments corresponding to Jα61 and Jα56 CEs are indicated (arrows). The fragment corresponding to the germline TCRα sJ allele is indicated by an asterisk (F). Retention of the Jα56 CE band in samples treated with mung bean nuclease and exonuclease V was quantified as described in Materials and methods (G).

    Journal: The Journal of Experimental Medicine

    Article Title: Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

    doi: 10.1084/jem.20061460

    Figure Lengend Snippet: Increased TCRα sJ locus CE accumulation in Atm −/− thymocytes. (A) Southern analysis strategy showing the relative positions of the NsiI (N) and StuI (S) sites, PS6, and the CαI probe. Fragment sizes generated by germline TCRα sJ alleles and Jα56 and Jα61 CEs are indicated. The schematics are not drawn to scale. (B) Southern blot analyses of total thymocyte DNA and kidney DNA (K) digested with either NsiI or HincII and hybridized to the CαI probe or PS6. Bands corresponding to Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline fragment is indicated by an asterisk. Hybridization of StuI and NsiI digested DNA to PR2 is shown as a DNA loading control. The molecular mass markers (in kb) are indicated. (C) LMPCR analysis of Jα56 and Jα61 CEs, as described in Materials and methods. Shown are fivefold serial dilutions of linker-ligated samples. IL-2 gene PCR is also shown as a DNA loading control. (D and E) Exonuclease V sensitivity of Jα56 CEs was assayed by hybridizing the membrane from Fig. 3D to the CαI probe. Bands corresponding to the Jα61 and Jα56 CEs are indicated (arrows). The band corresponding to the germline TCRα sJ allele is indicated by an asterisk. The positions of the molecular mass markers (in kb) are indicated (D). Retention of the Jα56 CE band in exonuclease V–treated samples was quantified as described in Materials and methods (E). (F and G) TCRα sJ/sJ :Atm − / − thymocyte DNA was treated with mung bean nuclease followed by exonuclease V before digestion with StuI and hybridization to the CαI probe, as described in Materials and methods. Fragments corresponding to Jα61 and Jα56 CEs are indicated (arrows). The fragment corresponding to the germline TCRα sJ allele is indicated by an asterisk (F). Retention of the Jα56 CE band in samples treated with mung bean nuclease and exonuclease V was quantified as described in Materials and methods (G).

    Article Snippet: For exonuclease V digestion, 15 μg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses.

    Techniques: Generated, Southern Blot, Hybridization, Polymerase Chain Reaction

    Equivalent TCRα sJ locus SE accumulation in Atm +/+ and Atm −/− thymocytes. (A) Schematics of the wild-type TCRα + and TCRα sJ loci. The Vα (open rectangles) and Jα (closed rectangles) gene segments are shown, as is the TCRα constant region gene (Cα; gray rectangles). The number in parenthesis indicates the number of Jα segments in the locus. (B) Southern blot analysis strategy. The relative positions of the HincII (H) and StuI (S) sites and P8 are shown. Fragment sizes generated by germline TCRα sJ alleles and Jα56 and Jα61 SEs are indicated. The schematics are not drawn to scale. (C) Southern blot analyses of total thymocyte DNA and kidney DNA (K) digested with either StuI or HincII and hybridized to P8. Hybridization to PR2 is shown as a DNA loading control. The fragment corresponding to the germline TCRα sJ allele is indicated by the asterisk. The fragments corresponding to the Jα61 SE are also indicated (arrows). The molecular mass markers (in kb) are indicated. (D and E) Thymocyte genomic DNA was digested in the absence (−) or in the presence of increasing concentrations of exonuclease V (ExoV) before digestions with StuI and hybridization to P8 (D). The fragment corresponding to the germline TCRα sJ allele is indicated by the asterisk. Retention of the Jα61 SE band (arrow) in exonuclease V–treated samples was quantified as described in Materials and methods (E). (F) LMPCR analysis of Jα56 and Jα61 SEs, as described in the Materials and methods. Shown are fivefold serial dilutions of linker-ligated samples, as well as the no-ligase control (−). IL-2 gene PCR is also shown as a DNA loading control.

    Journal: The Journal of Experimental Medicine

    Article Title: Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

    doi: 10.1084/jem.20061460

    Figure Lengend Snippet: Equivalent TCRα sJ locus SE accumulation in Atm +/+ and Atm −/− thymocytes. (A) Schematics of the wild-type TCRα + and TCRα sJ loci. The Vα (open rectangles) and Jα (closed rectangles) gene segments are shown, as is the TCRα constant region gene (Cα; gray rectangles). The number in parenthesis indicates the number of Jα segments in the locus. (B) Southern blot analysis strategy. The relative positions of the HincII (H) and StuI (S) sites and P8 are shown. Fragment sizes generated by germline TCRα sJ alleles and Jα56 and Jα61 SEs are indicated. The schematics are not drawn to scale. (C) Southern blot analyses of total thymocyte DNA and kidney DNA (K) digested with either StuI or HincII and hybridized to P8. Hybridization to PR2 is shown as a DNA loading control. The fragment corresponding to the germline TCRα sJ allele is indicated by the asterisk. The fragments corresponding to the Jα61 SE are also indicated (arrows). The molecular mass markers (in kb) are indicated. (D and E) Thymocyte genomic DNA was digested in the absence (−) or in the presence of increasing concentrations of exonuclease V (ExoV) before digestions with StuI and hybridization to P8 (D). The fragment corresponding to the germline TCRα sJ allele is indicated by the asterisk. Retention of the Jα61 SE band (arrow) in exonuclease V–treated samples was quantified as described in Materials and methods (E). (F) LMPCR analysis of Jα56 and Jα61 SEs, as described in the Materials and methods. Shown are fivefold serial dilutions of linker-ligated samples, as well as the no-ligase control (−). IL-2 gene PCR is also shown as a DNA loading control.

    Article Snippet: For exonuclease V digestion, 15 μg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses.

    Techniques: Southern Blot, Generated, Hybridization, Polymerase Chain Reaction

    S1 nuclease protection mapping of the ABCA2 transcription start site. A 5′-end phosphorylated 479 bp double-stranded DNA template for the S1 probe was generated by PCR and digested with λ exonuclease to generate an antisense single-stranded DNA probe that was end labeled with [γ- 32 P]ATP and hybridized to total RNA from BE(2)-M17 neuroblastoma cells. Following digestion with S1 nuclease, the protected fragment was separated by electrophoresis in a 6% polyacrylamide–7 M urea gel followed by autoradiography. The antisense strand of the 479 bp DNA template was manually sequenced and the φX174 Hin f DNA ladder was radiolabeled with [γ- 32 P]ATP to serve as sequence and size markers respectively. An arrow indicates a major protected fragment of 152 bp, located 95 bp upstream of the ATG translation start site that represents the major transcription start site.

    Journal: Nucleic Acids Research

    Article Title: Reciprocal regulation of expression of the human adenosine 5?-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors

    doi:

    Figure Lengend Snippet: S1 nuclease protection mapping of the ABCA2 transcription start site. A 5′-end phosphorylated 479 bp double-stranded DNA template for the S1 probe was generated by PCR and digested with λ exonuclease to generate an antisense single-stranded DNA probe that was end labeled with [γ- 32 P]ATP and hybridized to total RNA from BE(2)-M17 neuroblastoma cells. Following digestion with S1 nuclease, the protected fragment was separated by electrophoresis in a 6% polyacrylamide–7 M urea gel followed by autoradiography. The antisense strand of the 479 bp DNA template was manually sequenced and the φX174 Hin f DNA ladder was radiolabeled with [γ- 32 P]ATP to serve as sequence and size markers respectively. An arrow indicates a major protected fragment of 152 bp, located 95 bp upstream of the ATG translation start site that represents the major transcription start site.

    Article Snippet: Two micrograms of the PCR product was digested with λ exonuclease (Invitrogen) in 67 mM glycine–KOH pH 9.4, 2.5 mM MgCl2 , 50 µg/ml BSA at 37°C for 30 min to generate the antisense single-stranded DNA.

    Techniques: Generated, Polymerase Chain Reaction, Labeling, Electrophoresis, Autoradiography, Sequencing

    Schematic overview of the enzymatic steps of the probe generation protocol Templates were amplified from the template library pool by PCR using primers specific for each FISH genotyping round. Lambda exonuclease selectively digested the 5′‐phosphorylated strand, leaving only the 5′‐phosphorothioate strand. Fluorescently labeled and phosphorothioate‐modified elongation probes were hybridized to the ssDNA template and elongated with DreamTaq polymerase. The dsDNA product was digested with restriction enzyme SchI, removing the phosphorothioate bonds from the unlabeled strand. Lambda exonuclease digestion produced the final FISH probe.

    Journal: Molecular Systems Biology

    Article Title: In situ genotyping of a pooled strain library after characterizing complex phenotypes

    doi: 10.15252/msb.20177951

    Figure Lengend Snippet: Schematic overview of the enzymatic steps of the probe generation protocol Templates were amplified from the template library pool by PCR using primers specific for each FISH genotyping round. Lambda exonuclease selectively digested the 5′‐phosphorylated strand, leaving only the 5′‐phosphorothioate strand. Fluorescently labeled and phosphorothioate‐modified elongation probes were hybridized to the ssDNA template and elongated with DreamTaq polymerase. The dsDNA product was digested with restriction enzyme SchI, removing the phosphorothioate bonds from the unlabeled strand. Lambda exonuclease digestion produced the final FISH probe.

    Article Snippet: The phosphorylated strand was selectively digested by lambda exonuclease (Thermo Scientific) treatment for 30 min at 37°C followed by heat inactivation at 80°C for 10 min.

    Techniques: Amplification, Polymerase Chain Reaction, Fluorescence In Situ Hybridization, Labeling, Modification, Produced

    Products from the different steps of the FISH probe production protocol Products were run on a 10% polyacrylamide gel and imaged in (A) Cy3 and (B) Cy5 channels. (M): Cy3‐ and Cy5‐labeled 39‐nt and 19‐nt ssDNA probes were used as size references. (1) and (2): the initial fluorescent elongation product for the two rounds of FISH probe generation, respectively. (3) and (4): SchI digestion of the elongation products for rounds one and two, respectively. (5) and (6): Lambda exonuclease treatment and gel‐purified product for rounds one and two, respectively.

    Journal: Molecular Systems Biology

    Article Title: In situ genotyping of a pooled strain library after characterizing complex phenotypes

    doi: 10.15252/msb.20177951

    Figure Lengend Snippet: Products from the different steps of the FISH probe production protocol Products were run on a 10% polyacrylamide gel and imaged in (A) Cy3 and (B) Cy5 channels. (M): Cy3‐ and Cy5‐labeled 39‐nt and 19‐nt ssDNA probes were used as size references. (1) and (2): the initial fluorescent elongation product for the two rounds of FISH probe generation, respectively. (3) and (4): SchI digestion of the elongation products for rounds one and two, respectively. (5) and (6): Lambda exonuclease treatment and gel‐purified product for rounds one and two, respectively.

    Article Snippet: The phosphorylated strand was selectively digested by lambda exonuclease (Thermo Scientific) treatment for 30 min at 37°C followed by heat inactivation at 80°C for 10 min.

    Techniques: Fluorescence In Situ Hybridization, Labeling, Purification