rsai  (New England Biolabs)


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    Name:
    RsaI
    Description:
    RsaI 5 000 units
    Catalog Number:
    R0167L
    Price:
    249
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    New England Biolabs rsai
    RsaI
    RsaI 5 000 units
    https://www.bioz.com/result/rsai/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rsai - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA"

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.014

    Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).
    Figure Legend Snippet: Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).

    Techniques Used: Binding Assay, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Quantitation Assay, Flow Cytometry, Cytometry, Transfection, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Cotransfection

    2) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19139-4

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    3) Product Images from "Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes ▿"

    Article Title: Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes ▿

    Journal:

    doi: 10.1128/JCM.01550-08

    Real-time PCR assay for bla KPC . Real-time PCR using a TaqMan probe generates a 399-bp amplicon for all bla KPC genes. Amplicons from positive samples can be digested with BstNI and RsaI to detect the nucleotide polymorphisms in the PCR amplicon reported
    Figure Legend Snippet: Real-time PCR assay for bla KPC . Real-time PCR using a TaqMan probe generates a 399-bp amplicon for all bla KPC genes. Amplicons from positive samples can be digested with BstNI and RsaI to detect the nucleotide polymorphisms in the PCR amplicon reported

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

    4) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    5) Product Images from "Mobilization of LINE-1 in irradiated mammary gland tissue may potentially contribute to low dose radiation-induced genomic instability"

    Article Title: Mobilization of LINE-1 in irradiated mammary gland tissue may potentially contribute to low dose radiation-induced genomic instability

    Journal: Genes & Cancer

    doi:

    CpG methylation of LINE-1 promoter in the mammary gland of irradiated rats determined by the COBRA assay A. PCR amplification of the 163-bp from LINE-1 promoter. B. Methylation-dependent retention of pre-existing BstUI sites. Unmethylated CpG cytosines (highlighted) in the CGCG recognition sequence can be lost by bisulfite conversion, resulting in the uncut 163-bp fragments. Methylation at both sites allows the cleavage, resulting in 80/83-bp bands. C. Methylation-dependent retention of cytosine (highlighted) in the GGCACG sequence forms the RsaI recognition site, thus leading to the cleavage of the 163-bp fragment into 48- and 115-bp fragments. The loss of methylation at CpG cytosine sites will prevent the cleavage. D. Quantification of the cut BSTU1 fragments by AlphaView presented as mean values ± SD, n=4-6. * - significantly different from the respective control, p
    Figure Legend Snippet: CpG methylation of LINE-1 promoter in the mammary gland of irradiated rats determined by the COBRA assay A. PCR amplification of the 163-bp from LINE-1 promoter. B. Methylation-dependent retention of pre-existing BstUI sites. Unmethylated CpG cytosines (highlighted) in the CGCG recognition sequence can be lost by bisulfite conversion, resulting in the uncut 163-bp fragments. Methylation at both sites allows the cleavage, resulting in 80/83-bp bands. C. Methylation-dependent retention of cytosine (highlighted) in the GGCACG sequence forms the RsaI recognition site, thus leading to the cleavage of the 163-bp fragment into 48- and 115-bp fragments. The loss of methylation at CpG cytosine sites will prevent the cleavage. D. Quantification of the cut BSTU1 fragments by AlphaView presented as mean values ± SD, n=4-6. * - significantly different from the respective control, p

    Techniques Used: CpG Methylation Assay, Irradiation, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Amplification, Methylation, Sequencing

    A. Preparation of methylation gradient for testing primers for the COBRA assay. Steps of preparation of methylation gradients (0% - 100% methylation) by mixing fully unmethylated and fully methylated DNA. B. A methylation standard after the digestion of bisulfite treated DNA with BstUI and RsaI restriction endonucleases. The higher the percentage of the cut 163-bp fragment by BstUI, the higher the methylation status of DNA. The higher the percentage of the uncut 163-bp fragment, the lower the methylation status of DNA.
    Figure Legend Snippet: A. Preparation of methylation gradient for testing primers for the COBRA assay. Steps of preparation of methylation gradients (0% - 100% methylation) by mixing fully unmethylated and fully methylated DNA. B. A methylation standard after the digestion of bisulfite treated DNA with BstUI and RsaI restriction endonucleases. The higher the percentage of the cut 163-bp fragment by BstUI, the higher the methylation status of DNA. The higher the percentage of the uncut 163-bp fragment, the lower the methylation status of DNA.

    Techniques Used: Methylation, Combined Bisulfite Restriction Analysis Assay

    6) Product Images from "Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer"

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-28-125

    Imprinting analysis of LIT1 in gastric cancer . RsaI digestion of a 410 bp DNA PCR product (G1, G2) yielded bands of 222 and 188 bp indicating heterozygous specimens. RsaI digestion of RT-PCR amplification (Rn1, Rn2) showed only one allele expression in both normal tissues indicating maintenance of constitutional imprinting. Rt1, Rt2 displayed three bands in tumor specimens indicating loss of imprinting in contrast to their matching normal tissues (Rn1, Rn2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.
    Figure Legend Snippet: Imprinting analysis of LIT1 in gastric cancer . RsaI digestion of a 410 bp DNA PCR product (G1, G2) yielded bands of 222 and 188 bp indicating heterozygous specimens. RsaI digestion of RT-PCR amplification (Rn1, Rn2) showed only one allele expression in both normal tissues indicating maintenance of constitutional imprinting. Rt1, Rt2 displayed three bands in tumor specimens indicating loss of imprinting in contrast to their matching normal tissues (Rn1, Rn2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Marker

    Imprinting analysis of H19 in gastric cancer . H19 heterozygosity showed 655 bp DNA PCR product yielded bands of 487 and 168 bp by RsaI digestion (G1, G2). Normal tissues (N1, N2) showed only one allele expression indicating maintenance of normal imprinting (displayed 407 and 168 bp, 575 bp respectively by RsaI digestion RT-PCR products). T1, T2 displayed both three bands (575, 407 and 168 bp respectively) in tumor tissues indicating loss of imprinting in contrast to their matching normal tissues (N1, N2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.
    Figure Legend Snippet: Imprinting analysis of H19 in gastric cancer . H19 heterozygosity showed 655 bp DNA PCR product yielded bands of 487 and 168 bp by RsaI digestion (G1, G2). Normal tissues (N1, N2) showed only one allele expression indicating maintenance of normal imprinting (displayed 407 and 168 bp, 575 bp respectively by RsaI digestion RT-PCR products). T1, T2 displayed both three bands (575, 407 and 168 bp respectively) in tumor tissues indicating loss of imprinting in contrast to their matching normal tissues (N1, N2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.

    Techniques Used: Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker

    7) Product Images from "Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA"

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.05.014

    Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).
    Figure Legend Snippet: Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).

    Techniques Used: Binding Assay, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Quantitation Assay, Flow Cytometry, Cytometry, Transfection, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Cotransfection

    8) Product Images from "Impacts of Edaphic Factors on Communities of Ammonia-Oxidizing Archaea, Ammonia-Oxidizing Bacteria and Nitrification in Tropical Soils"

    Article Title: Impacts of Edaphic Factors on Communities of Ammonia-Oxidizing Archaea, Ammonia-Oxidizing Bacteria and Nitrification in Tropical Soils

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089568

    Non-metric multidimensional scaling plots of archaeal amoA terminal restriction fragment profiles generated by digestion with MspI (Panel A) or RsaI (Panel B). Symbol colors correspond to soil types, letters indicate soil names and values are the replicate number for the indicated soil. Soil name abbreviations: A, Arena; B, Brasso; E, Ecclesville; P, Piarco; R, River Estate; S, St. Augustine; T, Talparo; W, Princes Town.
    Figure Legend Snippet: Non-metric multidimensional scaling plots of archaeal amoA terminal restriction fragment profiles generated by digestion with MspI (Panel A) or RsaI (Panel B). Symbol colors correspond to soil types, letters indicate soil names and values are the replicate number for the indicated soil. Soil name abbreviations: A, Arena; B, Brasso; E, Ecclesville; P, Piarco; R, River Estate; S, St. Augustine; T, Talparo; W, Princes Town.

    Techniques Used: Generated

    Non-metric multidimensional scaling plots of bacterial amoA terminal restriction fragment profiles generated by digestion with MspI (Panel A) or RsaI (Panel B). Symbol colors correspond to soil types, letters indicate soil names and values are the replicate number from the indicated soil. Soil name abbreviations: A, Arena; B, Brasso; E, Ecclesville; P, Piarco; R, River Estate; S, St. Augustine; T, Talparo; W, Princes Town.
    Figure Legend Snippet: Non-metric multidimensional scaling plots of bacterial amoA terminal restriction fragment profiles generated by digestion with MspI (Panel A) or RsaI (Panel B). Symbol colors correspond to soil types, letters indicate soil names and values are the replicate number from the indicated soil. Soil name abbreviations: A, Arena; B, Brasso; E, Ecclesville; P, Piarco; R, River Estate; S, St. Augustine; T, Talparo; W, Princes Town.

    Techniques Used: Generated

    9) Product Images from "WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †"

    Article Title: WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01364-07

    Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Isolation, Transduction, Expressing, Hybridization

    TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Mutagenesis, Infection, Cell Culture, Transduction, Western Blot, Activity Assay, Staining, Standard Deviation, Quantitation Assay, Isolation, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation

    TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Transduction, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Generated, Lambda DNA Preparation, Plasmid Preparation

    Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Expressing, Over Expression, Isolation, Transduction, Plasmid Preparation, Variant Assay, Activity Assay, Hybridization

    10) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    11) Product Images from "Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona"

    Article Title: Isolation and Identification of Rickettsia massiliae from Rhipicephalus sanguineus Ticks Collected in Arizona

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00122-06

    RFLP typing of spotted fever group rickettsiae in R. sanguineus ticks. The 70- to 602-nucleotide fragment of the rOmpA gene was amplified using seminested PCR, followed by restriction enzyme digestion with RsaI (A) and PstI (B). Restriction patterns of
    Figure Legend Snippet: RFLP typing of spotted fever group rickettsiae in R. sanguineus ticks. The 70- to 602-nucleotide fragment of the rOmpA gene was amplified using seminested PCR, followed by restriction enzyme digestion with RsaI (A) and PstI (B). Restriction patterns of

    Techniques Used: Amplification, Polymerase Chain Reaction

    12) Product Images from "Detection of circular telomeric DNA without 2-D gel electrophoresis"

    Article Title: Detection of circular telomeric DNA without 2-D gel electrophoresis

    Journal: DNA and cell biology

    doi: 10.1089/dna.2008.0741

    Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior
    Figure Legend Snippet: Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior

    Techniques Used: Incubation, Hybridization

    Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound
    Figure Legend Snippet: Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound

    Techniques Used: Labeling

    The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part
    Figure Legend Snippet: The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis

    13) Product Images from "WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †"

    Article Title: WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01364-07

    Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Isolation, Transduction, Expressing, Hybridization

    TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Mutagenesis, Infection, Cell Culture, Transduction, Western Blot, Activity Assay, Staining, Standard Deviation, Quantitation Assay, Isolation, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation

    TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Transduction, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Generated, Lambda DNA Preparation, Plasmid Preparation

    Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Expressing, Over Expression, Isolation, Transduction, Plasmid Preparation, Variant Assay, Activity Assay, Hybridization

    14) Product Images from "The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions"

    Article Title: The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions

    Journal: bioRxiv

    doi: 10.1101/2019.12.11.873422

    Myc-Ku80 α5 mutant or Myc-Ku80 DEB expression compromises cell viability but does not lead to telomere shortening or t-circle formation in short term cultures. (A) Representative Southern blot analysis to measure telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku80 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku80 siRNA was used as positive and negative controls. KD indicates siRNA-mediated knockdown. Telomere Southern blots were quantified via TeloTool software and the red dot in each lane denotes mean telomere length. Additional representative experiment included in Supplemental Figure S3 (B) Same as A except telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku70 transgenes. Additional representative experiment included in Supplemental Figure S3 (C) 2D gel electrophoresis of HinfI/RsaI digested genomic DNA prepared from indicated cell lines that were subjected to either Ku70 or Ku80 knockdown. U2OS control cell line showing t-circles (D) Analysis of cell viability by Annexin V staining of indicated cell lines expressing Myc-Ku80 or Myc-Ku70 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku70 or Ku80 siRNA was used as controls.
    Figure Legend Snippet: Myc-Ku80 α5 mutant or Myc-Ku80 DEB expression compromises cell viability but does not lead to telomere shortening or t-circle formation in short term cultures. (A) Representative Southern blot analysis to measure telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku80 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku80 siRNA was used as positive and negative controls. KD indicates siRNA-mediated knockdown. Telomere Southern blots were quantified via TeloTool software and the red dot in each lane denotes mean telomere length. Additional representative experiment included in Supplemental Figure S3 (B) Same as A except telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku70 transgenes. Additional representative experiment included in Supplemental Figure S3 (C) 2D gel electrophoresis of HinfI/RsaI digested genomic DNA prepared from indicated cell lines that were subjected to either Ku70 or Ku80 knockdown. U2OS control cell line showing t-circles (D) Analysis of cell viability by Annexin V staining of indicated cell lines expressing Myc-Ku80 or Myc-Ku70 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku70 or Ku80 siRNA was used as controls.

    Techniques Used: Mutagenesis, Expressing, Southern Blot, Plasmid Preparation, Transfection, Software, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining

    15) Product Images from "Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿"

    Article Title: Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00920-07

    RFLP analysis of espP . PCR products of 3,760 bp obtained with primers espPlong-1 and espPlong-2 were digested with RsaI or SspI and separated using agarose gel electrophoresis. Lanes 1 and 2, RsaI restriction patterns R1 and R2, respectively; lanes 3
    Figure Legend Snippet: RFLP analysis of espP . PCR products of 3,760 bp obtained with primers espPlong-1 and espPlong-2 were digested with RsaI or SspI and separated using agarose gel electrophoresis. Lanes 1 and 2, RsaI restriction patterns R1 and R2, respectively; lanes 3

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    16) Product Images from "Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells *Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells * S⃞"

    Article Title: Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells *Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M802942200

    Selected areas of DNA methylation in control or arsenic-exposed RWPE-1 cells at 4 weeks of exposure using arbitrarily primed PCR after restriction enzyme digestions. Shown are details of a representative gel with an area of distinct DNA hypomethylation in arsenic-treated DNA ( left panels, two-headed arrows ) and densitometric analysis ( n = 3) of this band ( right panel ). See methods for details. An asterisk indicates a significant difference ( p ≤ 0.05) from control. Unmethylated DNA is not protected from digestion by methylation-sensitive HpaII restriction enzyme resulting in a loss of DNA for PCR amplification. Digestion with RsaI + MspI would also result in no PCR amplification regardless of methylation status. Digestion with RsaI alone and RsaI + MspI were used as controls to determine whether there was differential methylation.
    Figure Legend Snippet: Selected areas of DNA methylation in control or arsenic-exposed RWPE-1 cells at 4 weeks of exposure using arbitrarily primed PCR after restriction enzyme digestions. Shown are details of a representative gel with an area of distinct DNA hypomethylation in arsenic-treated DNA ( left panels, two-headed arrows ) and densitometric analysis ( n = 3) of this band ( right panel ). See methods for details. An asterisk indicates a significant difference ( p ≤ 0.05) from control. Unmethylated DNA is not protected from digestion by methylation-sensitive HpaII restriction enzyme resulting in a loss of DNA for PCR amplification. Digestion with RsaI + MspI would also result in no PCR amplification regardless of methylation status. Digestion with RsaI alone and RsaI + MspI were used as controls to determine whether there was differential methylation.

    Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Amplification

    17) Product Images from "Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells *Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells * S⃞"

    Article Title: Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells *Interplay between Cellular Methyl Metabolism and Adaptive Efflux during Oncogenic Transformation from Chronic Arsenic Exposure in Human Cells * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M802942200

    Selected areas of DNA methylation in control or arsenic-exposed RWPE-1 cells at 4 weeks of exposure using arbitrarily primed PCR after restriction enzyme digestions. Shown are details of a representative gel with an area of distinct DNA hypomethylation in arsenic-treated DNA ( left panels, two-headed arrows ) and densitometric analysis ( n = 3) of this band ( right panel ). See methods for details. An asterisk indicates a significant difference ( p ≤ 0.05) from control. Unmethylated DNA is not protected from digestion by methylation-sensitive HpaII restriction enzyme resulting in a loss of DNA for PCR amplification. Digestion with RsaI + MspI would also result in no PCR amplification regardless of methylation status. Digestion with RsaI alone and RsaI + MspI were used as controls to determine whether there was differential methylation.
    Figure Legend Snippet: Selected areas of DNA methylation in control or arsenic-exposed RWPE-1 cells at 4 weeks of exposure using arbitrarily primed PCR after restriction enzyme digestions. Shown are details of a representative gel with an area of distinct DNA hypomethylation in arsenic-treated DNA ( left panels, two-headed arrows ) and densitometric analysis ( n = 3) of this band ( right panel ). See methods for details. An asterisk indicates a significant difference ( p ≤ 0.05) from control. Unmethylated DNA is not protected from digestion by methylation-sensitive HpaII restriction enzyme resulting in a loss of DNA for PCR amplification. Digestion with RsaI + MspI would also result in no PCR amplification regardless of methylation status. Digestion with RsaI alone and RsaI + MspI were used as controls to determine whether there was differential methylation.

    Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Amplification

    18) Product Images from "Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters"

    Article Title: Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters

    Journal: Environmental Microbiology

    doi: 10.1111/j.1462-2920.2008.01793.x

    Self-organizing tree (SOTA) of terminal restriction fragment length polymorphism (T-RFLP) profiles emerging from the 18S rRNA sequences from the different experimental treatments. Samples digested (A) with HhaI and (B) with RsaI. Time 0: T-RFLP profile of rRNA from the entire eukaryotic community at the beginning of the experiment. Unlabelled Pro and Unlabelled Syn: T-RFLP profiles of the unlabelled eukaryotic rRNA, collected from the density gradient, from the bottles that were incubated with labelled Prochlorococcus and Synechococcus respectively. Labelled Pro and Labelled Syn : T-RFLP profiles of the heavily labelled eukaryotic rRNA that was collected from the same gradient. A and B next to the data points represent the two biological replicates.
    Figure Legend Snippet: Self-organizing tree (SOTA) of terminal restriction fragment length polymorphism (T-RFLP) profiles emerging from the 18S rRNA sequences from the different experimental treatments. Samples digested (A) with HhaI and (B) with RsaI. Time 0: T-RFLP profile of rRNA from the entire eukaryotic community at the beginning of the experiment. Unlabelled Pro and Unlabelled Syn: T-RFLP profiles of the unlabelled eukaryotic rRNA, collected from the density gradient, from the bottles that were incubated with labelled Prochlorococcus and Synechococcus respectively. Labelled Pro and Labelled Syn : T-RFLP profiles of the heavily labelled eukaryotic rRNA that was collected from the same gradient. A and B next to the data points represent the two biological replicates.

    Techniques Used: Terminal Restriction Fragment Length Polymorphism, Incubation

    19) Product Images from "Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii"

    Article Title: Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-7-25

    Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)
    Figure Legend Snippet: Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    20) Product Images from "Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei"

    Article Title: Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh991

    Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.
    Figure Legend Snippet: Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.

    Techniques Used: Knock-Out, Agarose Gel Electrophoresis

    21) Product Images from "Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains ▿"

    Article Title: Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01336-08

    16S-23S ITS-PCR-RFLP patterns of Pectobacterium strains. The 16S-23S ITS region was amplified with primers L1 and G1, and the amplified DNA was digested with RsaI and then analyzed by gel electrophoresis. The strains used are indicated above the lanes, and strains are grouped into the clades defined by the MLSA. dH 2 O, distilled water.
    Figure Legend Snippet: 16S-23S ITS-PCR-RFLP patterns of Pectobacterium strains. The 16S-23S ITS region was amplified with primers L1 and G1, and the amplified DNA was digested with RsaI and then analyzed by gel electrophoresis. The strains used are indicated above the lanes, and strains are grouped into the clades defined by the MLSA. dH 2 O, distilled water.

    Techniques Used: Polymerase Chain Reaction, Amplification, Nucleic Acid Electrophoresis

    22) Product Images from "Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer"

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-28-125

    Imprinting analysis of LIT1 in gastric cancer . RsaI digestion of a 410 bp DNA PCR product (G1, G2) yielded bands of 222 and 188 bp indicating heterozygous specimens. RsaI digestion of RT-PCR amplification (Rn1, Rn2) showed only one allele expression in both normal tissues indicating maintenance of constitutional imprinting. Rt1, Rt2 displayed three bands in tumor specimens indicating loss of imprinting in contrast to their matching normal tissues (Rn1, Rn2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.
    Figure Legend Snippet: Imprinting analysis of LIT1 in gastric cancer . RsaI digestion of a 410 bp DNA PCR product (G1, G2) yielded bands of 222 and 188 bp indicating heterozygous specimens. RsaI digestion of RT-PCR amplification (Rn1, Rn2) showed only one allele expression in both normal tissues indicating maintenance of constitutional imprinting. Rt1, Rt2 displayed three bands in tumor specimens indicating loss of imprinting in contrast to their matching normal tissues (Rn1, Rn2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.

    Techniques Used: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Marker

    Imprinting analysis of H19 in gastric cancer . H19 heterozygosity showed 655 bp DNA PCR product yielded bands of 487 and 168 bp by RsaI digestion (G1, G2). Normal tissues (N1, N2) showed only one allele expression indicating maintenance of normal imprinting (displayed 407 and 168 bp, 575 bp respectively by RsaI digestion RT-PCR products). T1, T2 displayed both three bands (575, 407 and 168 bp respectively) in tumor tissues indicating loss of imprinting in contrast to their matching normal tissues (N1, N2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.
    Figure Legend Snippet: Imprinting analysis of H19 in gastric cancer . H19 heterozygosity showed 655 bp DNA PCR product yielded bands of 487 and 168 bp by RsaI digestion (G1, G2). Normal tissues (N1, N2) showed only one allele expression indicating maintenance of normal imprinting (displayed 407 and 168 bp, 575 bp respectively by RsaI digestion RT-PCR products). T1, T2 displayed both three bands (575, 407 and 168 bp respectively) in tumor tissues indicating loss of imprinting in contrast to their matching normal tissues (N1, N2). M, marker DL2000. Nc1, Nc2 represented RT-PCR without reverse transcriptase.

    Techniques Used: Polymerase Chain Reaction, Expressing, Reverse Transcription Polymerase Chain Reaction, Marker

    23) Product Images from "Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Gene Polymorphisms and Hepatitis B Virus Infection"

    Article Title: Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Gene Polymorphisms and Hepatitis B Virus Infection

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.23578

    RsaI Enzyme Digestion Products of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Gene at Position 1525 Lane 1, DNA marker (100 bp); lane 2, PCR product; lanes 3 and 5, AG genotype; lane 4, AA genotype; lane 6, GG genotype.
    Figure Legend Snippet: RsaI Enzyme Digestion Products of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Gene at Position 1525 Lane 1, DNA marker (100 bp); lane 2, PCR product; lanes 3 and 5, AG genotype; lane 4, AA genotype; lane 6, GG genotype.

    Techniques Used: Marker, Polymerase Chain Reaction

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Molecular Characterization and Phylogeny of a Phytoplasma Associated with Phyllody Disease of toria (Brassica rapa L. subsp. dichotoma (Roxb.)) in India
    Article Snippet: The PCR products of 16S rDNA (using R16F2n/R16R2 primer pair) and 23S rDNA (using P23S5F3/A23S3R3 primer pair) were purified by QIAquick PCR purification kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instruction. .. A total volume of 7 μl of PCR products were digested with restriction endonucleases Alu I, Hae III, Hha I, Hin fI, Mse I and Rsa I (New England BioLabs, Waverley, MA, USA) at 37°C for 3 h. Resultant restriction fragments were visualized by electrophoresis through 2.8% agarose gel with Tris–EDTA as running buffer. .. DNA fragment profiles in gels were visualized and recorded in gel documentation unit (XR documentation system, Bio-Rad, USA).

    Article Title: Polymorphism of the Prolactin Gene and Its Relationship with Milk Production in Gir and Kankrej Cattle
    Article Snippet: The PCR reaction products was electrophoresed on 1.5% agarose gel and stained with ethidium bromide to detect the amplification success. .. The PCR products were digested with 5 units of Rsa I (New England Biolabs) at 37°C for 1 h in a final reaction volume of 25 μl. .. After restriction digestion, the restricted fragments were analyzed electrophoretically using 3% agarose gel, stained with ethidium bromide.

    Electrophoresis:

    Article Title: Molecular Characterization and Phylogeny of a Phytoplasma Associated with Phyllody Disease of toria (Brassica rapa L. subsp. dichotoma (Roxb.)) in India
    Article Snippet: The PCR products of 16S rDNA (using R16F2n/R16R2 primer pair) and 23S rDNA (using P23S5F3/A23S3R3 primer pair) were purified by QIAquick PCR purification kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instruction. .. A total volume of 7 μl of PCR products were digested with restriction endonucleases Alu I, Hae III, Hha I, Hin fI, Mse I and Rsa I (New England BioLabs, Waverley, MA, USA) at 37°C for 3 h. Resultant restriction fragments were visualized by electrophoresis through 2.8% agarose gel with Tris–EDTA as running buffer. .. DNA fragment profiles in gels were visualized and recorded in gel documentation unit (XR documentation system, Bio-Rad, USA).

    Agarose Gel Electrophoresis:

    Article Title: Molecular Characterization and Phylogeny of a Phytoplasma Associated with Phyllody Disease of toria (Brassica rapa L. subsp. dichotoma (Roxb.)) in India
    Article Snippet: The PCR products of 16S rDNA (using R16F2n/R16R2 primer pair) and 23S rDNA (using P23S5F3/A23S3R3 primer pair) were purified by QIAquick PCR purification kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instruction. .. A total volume of 7 μl of PCR products were digested with restriction endonucleases Alu I, Hae III, Hha I, Hin fI, Mse I and Rsa I (New England BioLabs, Waverley, MA, USA) at 37°C for 3 h. Resultant restriction fragments were visualized by electrophoresis through 2.8% agarose gel with Tris–EDTA as running buffer. .. DNA fragment profiles in gels were visualized and recorded in gel documentation unit (XR documentation system, Bio-Rad, USA).

    Article Title: Telomere damage induces internal loops that generate telomeric circles
    Article Snippet: The digestion was precipitated and loaded on a sucrose gradient, 10%–20%–30% sucrose, 8 ml each fraction, in TNE buffer and centrifuged in SW32-Ti rotor (Beckman) at 30100 rpm (111265 g) for 16 h. The HMW fractions containing the telomeric repeats were collected, concentrated, and washed twice with Tris 10 mM pH 8.0 in Amicon Ultra-15 Ultracel-PL PLTK, 30 kDa MWCO (Millipore/MERCK UFC903024) filters. .. The DNA was then digested overnight with 50 units each of RsaI, AluI, MboI, HinfI, MspI, HphI, MnlI (NEB), and then separated on a 0.7% low-melting agarose gel (SeaPlaque Agarose, Lonza, 50100), without ethidium bromide. .. Fragments migrating above the 5 kb band of the marker were extracted using the Silica Bead DNA gel extraction kit (Thermo Fisher Scientific, K0513) following the manufacturer’s instructions, except that once the DNA was bound, the beads were not resuspended to avoid mechanical shearing of the DNA.

    Article Title: Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes ▿
    Article Snippet: Amplification was performed using recombinant Taq Polymerase (Qiagen) at a final magnesium concentration of 2 mM. .. The following cycling conditions were used: 95o for 2 min, followed by 35 cycles of 94°C for 2 s, 62°C for 10 s, and 72°C for 15 s. The amplicons from KPC-positive samples were then digested at 48°C for 1 h in RsaI and BstNI (New England Biolabs, Ipswitch, MA) using NEB buffer 2 (New England Biolabs) and electrophoresed on a 2% agarose gel to differentiate bla KPC-1 , bla KPC-2 , and bla KPC-3 . ..

    Amplification:

    Article Title: Assessment of Microbial Diversity in Four Southwestern United States Soils by 16S rRNA Gene Terminal Restriction Fragment Analysis
    Article Snippet: .. Fluorescent amplification products were ethanol precipitated and resuspended in 25 μl of sterile, distilled water, and 8 μl was digested with 5 U of Rsa I (New England Biolabs, Beverly, Mass.) in 12-μl reaction mixtures. .. Following restriction digestion, 1 μl of each digest was dried, suspended in 1.75 μl of loading buffer containing 0.25 μl of Genescan 2500 TAMRA size standard (ABI), a 5:1 mixture of deionized formamide-blue dextran, and 25 mM EDTA, and then denatured at 94°C for 2 min. Fragments were separated by electrophoresis in denaturing 4% polyacrylamide gels with an ABI 377 DNA sequencer.

    Purification:

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA
    Article Snippet: Gel pieces were then column purified using NucleoSpin. .. The purified fragment was digested with the blunt-end-generating restriction enzymes DraI and RsaI in CutSmart Buffer (New England Biolabs) at 37°C for 2 hr then column purified with NucleoSpin. .. A heterogeneous pool of the digested target region was ligated to the blunt-digested reporter using T4 Ligase (New England Biolabs) with an overnight incubation at 16°C.

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    Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of <t>RsaI</t> (R) and <t>DraI</t> (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).
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    Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Spliceosome-Mediated Pre-mRNA trans-Splicing Can Repair CEP290 mRNA

    doi: 10.1016/j.omtn.2018.05.014

    Figure Lengend Snippet: Identification of a Candidate Binding Domain to Target trans -Splicing Molecules to CEP290 Intron X-27 (A) Schematic of trans -splicing between a 5′ binding domain (BD) test PTM encoding the 5′ portion of GFP (5′ GFP) and a mini-gene target encoding the 3′ portion of GFP (3′ GFP). Watson-Crick base pairing is indicated by vertical dashed lines. Trans -splicing between the two pre-mRNAs results in reconstitution and expression of GFP. (B) Diagram of RsaI (R) and DraI (D) restriction sites within a region of CEP290 intron X-27. (C) Agarose gel electrophoresis after restriction enzyme digestion of a PCR fragment corresponding to the region described in (B). The fragment was amplified from genomic DNA, digested with restriction enzymes RsaI and DraI, and visualized on a 2% TBE-agarose gel. The fragment library numbers of visible bands of expected sizes are indicated according to the table of predicted fragments. (D) Quantitation by flow cytometry of GFP expression in HEK293T transiently transfected with plasmids encoding a fragment library test PTM (gray bars) or co-transfected with a test PTM and the 3′ GFP target (black bars). Samples with “f’ demark forward orientation that is not predicted to confer trans -splicing specificity; however, BD_05f did yield a slight improvement to GFP expression over no binding domain (NBD). (E) Agarose gel electrophoresis following RT-PCR using cDNA generated from HEK293T cells transfected with plasmids in (D). Primers were designed to specifically bind to the 5′ or 3′ portions of the GFP coding DNA sequence to validate trans -splicing between the 5′ test PTM and the 3′ GFP target pre-mRNAs. Data for the other test PTMs has been removed at the break indicated. Interestingly, untargeted PTM (NBD) also resulted in trans -splicing of RNA in agreement with observed GFP expression. MG, mini-gene. (F) Samples from (E) with primers designed to specifically bind to the 5′ portion of GFP or to exon 27 of Homo sapiens CEP290 . Image has been contrast enhanced to visualize the faint band in lane 7 for co-transfection of BD_07 with target mini-gene (MG).

    Article Snippet: The purified fragment was digested with the blunt-end-generating restriction enzymes DraI and RsaI in CutSmart Buffer (New England Biolabs) at 37°C for 2 hr then column purified with NucleoSpin.

    Techniques: Binding Assay, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Quantitation Assay, Flow Cytometry, Cytometry, Transfection, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Cotransfection

    Electrophoresis in 8% polyacrylamide gel of exon 2 amplification products of gene BoLA-DRB3 digested by endonucleases Rsa I (b), Hae III (a), and BstYI (c). Msp I fragments of plasmid pUC19 are used as a molecular marker. The length of fragments composing Rsa I, Hae III, or Bst YI DNA patterns is shown on pictures.

    Journal: The Scientific World Journal

    Article Title: Distribution of BoLA-DRB3 Allelic Frequencies and Identification of Two New Alleles in Iranian Buffalo Breed

    doi: 10.1100/2012/863024

    Figure Lengend Snippet: Electrophoresis in 8% polyacrylamide gel of exon 2 amplification products of gene BoLA-DRB3 digested by endonucleases Rsa I (b), Hae III (a), and BstYI (c). Msp I fragments of plasmid pUC19 are used as a molecular marker. The length of fragments composing Rsa I, Hae III, or Bst YI DNA patterns is shown on pictures.

    Article Snippet: Samples were digested with Rsa I or Hae III (20 units, New England Biolabs) for 2 h at 37°C in a total volume of 20 μ L. Samples were digested with BstYI (20 units, New England Biolabs) for 2 h at 50°C followed by 15 min at 85°C in a total volume of 20 μ L.

    Techniques: Electrophoresis, Amplification, Plasmid Preparation, Marker

    A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Journal: Nature Communications

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1038/s41467-020-19139-4

    Figure Lengend Snippet: A two-step procedure for the purification of mammalian telomeres. a Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk genomic DNA in a sucrose gradient. Genomic DNA (∼2.5 mg) from SV40LT-immortalized MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (∼1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. Source data are provided as a Source Data File. b Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (a), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI, and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (∼1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. Source data are provided as a Source Data File. c Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed L1 repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. Source data are provided as a Source Data File. d Single-molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Article Snippet: The DNA was then digested overnight with 50 units each of RsaI, AluI, MboI, HinfI, MspI, HphI, MnlI (NEB), and then separated on a 0.7% low-melting agarose gel (SeaPlaque Agarose, Lonza, 50100), without ethidium bromide.

    Techniques: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    Real-time PCR assay for bla KPC . Real-time PCR using a TaqMan probe generates a 399-bp amplicon for all bla KPC genes. Amplicons from positive samples can be digested with BstNI and RsaI to detect the nucleotide polymorphisms in the PCR amplicon reported

    Journal:

    Article Title: Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes ▿

    doi: 10.1128/JCM.01550-08

    Figure Lengend Snippet: Real-time PCR assay for bla KPC . Real-time PCR using a TaqMan probe generates a 399-bp amplicon for all bla KPC genes. Amplicons from positive samples can be digested with BstNI and RsaI to detect the nucleotide polymorphisms in the PCR amplicon reported

    Article Snippet: The following cycling conditions were used: 95o for 2 min, followed by 35 cycles of 94°C for 2 s, 62°C for 10 s, and 72°C for 15 s. The amplicons from KPC-positive samples were then digested at 48°C for 1 h in RsaI and BstNI (New England Biolabs, Ipswitch, MA) using NEB buffer 2 (New England Biolabs) and electrophoresed on a 2% agarose gel to differentiate bla KPC-1 , bla KPC-2 , and bla KPC-3 .

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction