hinfi  (New England Biolabs)


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    HinfI
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    HinfI 25 000 units
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    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs hinfi
    HinfI
    HinfI 25 000 units
    https://www.bioz.com/result/hinfi/product/New England Biolabs
    Average 95 stars, based on 59 article reviews
    Price from $9.99 to $1999.99
    hinfi - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    2) 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 IGF2 in gastric cancer . DNA (G1) and RT-PCR (G3) amplification using primers P1 and P3 and DNA amplification by PCR with primers P2 and P3 (G2) represented 1.4 kb, 1.12 kb and 292 bp respectively (see details in methods section). G1, G2 and G3 are PCR products of the same normal tissue. ApaI- and HinfI-digested normal tissue DNA PCR (Gn) from primers P2 and P3 displayed two bands of 256 and 231 bp indicating heterozygosity. The digested nested PCR product from primers P2 and P3 using the 1.12 kb RT-PCR product as a template showed monoallelic expression of IGF2 in normal (Rn1, Rn2) and biallelic expression in tumor (Rt1, Rt2) tissues.
    Figure Legend Snippet: Imprinting analysis of IGF2 in gastric cancer . DNA (G1) and RT-PCR (G3) amplification using primers P1 and P3 and DNA amplification by PCR with primers P2 and P3 (G2) represented 1.4 kb, 1.12 kb and 292 bp respectively (see details in methods section). G1, G2 and G3 are PCR products of the same normal tissue. ApaI- and HinfI-digested normal tissue DNA PCR (Gn) from primers P2 and P3 displayed two bands of 256 and 231 bp indicating heterozygosity. The digested nested PCR product from primers P2 and P3 using the 1.12 kb RT-PCR product as a template showed monoallelic expression of IGF2 in normal (Rn1, Rn2) and biallelic expression in tumor (Rt1, Rt2) tissues.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Nested PCR, Expressing

    3) Product Images from "The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner"

    Article Title: The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr768

    Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.
    Figure Legend Snippet: Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.

    Techniques Used: Methylation, Plasmid Preparation, Sequencing, Activity Assay

    CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.
    Figure Legend Snippet: CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.

    Techniques Used: Methylation, Plasmid Preparation, Marker, Molecular Weight

    CcrM processivity assays using the 129-mer DNA as substrate ( Figure 2 C) and conducted in the absence ( A ) and with the addition ( B ) of competitor DNA. The expected running distance of partially protected intermediates is indicated on the right by asterisk. Undigested 129-mer DNA was used as control, indicating the expected running distance of fully protected DNA. The marker lane (indicated by M) contained PCR marker provided by New England Biolabs. The sizes of the bands are indicated on the left. ( A ) Initially all of the DNA is efficiently digested by HinfI (time point 0.1 min). Increasing protection from HinfI digestion is established over time, with nearly complete protection being achieved after 120 min. The 96 bp intermediate is present in large amounts, as well as low levels of the 54 bp intermediate, indicating distributive methylation by CcrM, as well as a preference for one target sequence over the other. ( B ) Competitor DNA is added after 3 min (indicated by the arrow). The protection state of the 129-mer substrate remains the same after supplementation with competitor, suggesting dissociation of CcrM from incompletely methylated DNA. The weak band appearing in the last lanes at low molecular weights corresponds to the 23-mer competitor which has become methylated and, thereby, protected against HinfI cleavage.
    Figure Legend Snippet: CcrM processivity assays using the 129-mer DNA as substrate ( Figure 2 C) and conducted in the absence ( A ) and with the addition ( B ) of competitor DNA. The expected running distance of partially protected intermediates is indicated on the right by asterisk. Undigested 129-mer DNA was used as control, indicating the expected running distance of fully protected DNA. The marker lane (indicated by M) contained PCR marker provided by New England Biolabs. The sizes of the bands are indicated on the left. ( A ) Initially all of the DNA is efficiently digested by HinfI (time point 0.1 min). Increasing protection from HinfI digestion is established over time, with nearly complete protection being achieved after 120 min. The 96 bp intermediate is present in large amounts, as well as low levels of the 54 bp intermediate, indicating distributive methylation by CcrM, as well as a preference for one target sequence over the other. ( B ) Competitor DNA is added after 3 min (indicated by the arrow). The protection state of the 129-mer substrate remains the same after supplementation with competitor, suggesting dissociation of CcrM from incompletely methylated DNA. The weak band appearing in the last lanes at low molecular weights corresponds to the 23-mer competitor which has become methylated and, thereby, protected against HinfI cleavage.

    Techniques Used: Marker, Polymerase Chain Reaction, Methylation, Sequencing

    4) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

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

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

    Journal: Nucleic Acids Research

    doi:

    Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).
    Figure Legend Snippet: Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).

    Techniques Used: Ligation, Polymerase Chain Reaction, Construct

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

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

    6) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    7) Product Images from "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1"

    Article Title: Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800121

    Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.
    Figure Legend Snippet: Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.

    Techniques Used: Transfection, Agarose Gel Electrophoresis, Amplification, Positive Control

    8) 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

    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 "Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells"

    Article Title: Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089546

    Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.
    Figure Legend Snippet: Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced

    11) Product Images from "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1"

    Article Title: Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800121

    Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.
    Figure Legend Snippet: Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.

    Techniques Used: Transfection, Agarose Gel Electrophoresis, Amplification, Positive Control

    12) Product Images from "Discovery of DNA methylation markers in cervical cancer using relaxation ranking"

    Article Title: Discovery of DNA methylation markers in cervical cancer using relaxation ranking

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-1-57

    Representative COBRA on 3 gene promoters ( SST , AUTS2 and SYCP3 ) . A: schematic representation of of the restriction enzyme sites in the virtual hypermethylated BSP nucleotide sequence after bisulfite treatment.(B: BstUI , T: TaqI and H: HinfI ). Bars represent CG site and arrow is TSS (retrieved from Ensembl). B: Result of COBRA analysis of BSP products of tumour samples (T1-T10) and 5 normal cervices (N1-N5), in vitro methylated DNA as a positive control (IV) and leukocyte DNA as a negative (unmethylated) control (L); lane B is water blank.
    Figure Legend Snippet: Representative COBRA on 3 gene promoters ( SST , AUTS2 and SYCP3 ) . A: schematic representation of of the restriction enzyme sites in the virtual hypermethylated BSP nucleotide sequence after bisulfite treatment.(B: BstUI , T: TaqI and H: HinfI ). Bars represent CG site and arrow is TSS (retrieved from Ensembl). B: Result of COBRA analysis of BSP products of tumour samples (T1-T10) and 5 normal cervices (N1-N5), in vitro methylated DNA as a positive control (IV) and leukocyte DNA as a negative (unmethylated) control (L); lane B is water blank.

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, Sequencing, In Vitro, Methylation, Positive Control

    13) 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

    14) Product Images from "Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia"

    Article Title: Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia

    Journal: Genes and immunity

    doi: 10.1038/gene.2012.40

    a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).
    Figure Legend Snippet: a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).

    Techniques Used: Molecular Weight, Marker, Polymerase Chain Reaction, Methylation, Whisker Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

    15) Product Images from "Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops"

    Article Title: Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9948-9957.2004

    Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.
    Figure Legend Snippet: Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.

    Techniques Used: Activity Assay, Isolation, Hybridization, TRAP Assay, Polymerase Chain Reaction, Fractionation, Quantitation Assay, Dot Blot, Plasmid Preparation, Clone Assay

    2D PFGE of TRFs from ALT and non-ALT cells. Total DNA (20 μg) was digested with HinfI/HaeIII and then separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe and was visualized using a PhosphorImager. The black and white arrows indicate linear- and circular-form DNA, respectively.
    Figure Legend Snippet: 2D PFGE of TRFs from ALT and non-ALT cells. Total DNA (20 μg) was digested with HinfI/HaeIII and then separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe and was visualized using a PhosphorImager. The black and white arrows indicate linear- and circular-form DNA, respectively.

    Techniques Used: Hybridization

    16) Product Images from "Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology"

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    Journal: Nature protocols

    doi: 10.1038/nprot.2018.012

    Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.
    Figure Legend Snippet: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Techniques Used: CRISPR, Non-Homologous End Joining, Mutagenesis, Mouse Assay, Generated, Electroporation

    17) 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

    18) Product Images from "GyrA ser83 and ParC trp106 Mutations in Salmonella enterica Serovar Typhi Isolated from Typhoid Fever Patients in Tertiary Care Hospital"

    Article Title: GyrA ser83 and ParC trp106 Mutations in Salmonella enterica Serovar Typhi Isolated from Typhoid Fever Patients in Tertiary Care Hospital

    Journal: Journal of Clinical and Diagnostic Research : JCDR

    doi: 10.7860/JCDR/2016/17677.8153

    Representative RFLP electrophoretic gel showing the gyrA PCR product digested with HinfI.
    Figure Legend Snippet: Representative RFLP electrophoretic gel showing the gyrA PCR product digested with HinfI.

    Techniques Used: Polymerase Chain Reaction

    19) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    20) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

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    Amplification:

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    Synthesized:

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    Neutralization:

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    Terminal Restriction Fragment Length Polymorphism:

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    Enzyme-linked Immunosorbent Assay:

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    Incubation:

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    TRF Assay:

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    Hybridization:

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    Southern Blot:

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    RLGS:

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    Imaging:

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    Sequencing:

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    Cellular Antioxidant Activity Assay:

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    Pulsed-Field Gel:

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    Article Snippet: .. Five micrograms of DNA were digested with Hinf I (10 U/μl, New England Biolabs, Ipswich, MA) and resolved on a 1.2% agarose gel by pulsed field gel electrophoresis (5 V/cm, 0.5–5 s switch times, 14 °C for 17 h). .. Lambda DNA‐Hind III digest (Bio‐Rad Laboratories, Segrate, Italy) was used for size determination of telomere restriction fragments (TRF).

    DNA Extraction:

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    Marker:

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    Methylation:

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    Mutagenesis:

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    Isolation:

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    Size-exclusion Chromatography:

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    Labeling:

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    Purification:

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    Article Snippet: .. Reconstituted DNA was digested with Hinf I and Rsa I (New England Biolabs) and again purified by phenol:chloroform extraction. .. For TRF analysis, 2 μg of digested DNA were separated on 0.6% agarose gels, which were vacuum-dried at 50 °C for 50 min. Gels were hybridized at 50 °C overnight with telomeric oligonucleotide probes (5′-(TTAGGG)5 −3′ or 5′-(CCCTAA)5 −3′), 5′-end labeled with T4 polynucleotide kinase (New England Biolabs) and [γ-32 P]ATP.

    Article Title: Epidemiological and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from the Ear Discharge of Outpatients with Chronic Otitis Media
    Article Snippet: .. PCR products were purified by the QIAquick-spin PCR purification kit (Qiagen Inc., Valencia, CA, U.S.A.) and followed by digestion with Hinf I and Bsm I (New England Biolabs Inc., Ipswich, MA, U.S.A.). .. The RFLP patterns were analyzed using 2% agarose gel electrophoresis.

    Polymerase Chain Reaction:

    Article Title: Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii
    Article Snippet: .. PCR products (15 μl) were partitioned into eight aliquots and separately digested overnight with 5 U of Hae III, Hha I, Alu I, Mbo I, Msp I, Rsa I, Hinf I and BstU I (New England Biolabs, Ipswich, MA, USA) in a total volume of 30 μl at 37 °C, with the exception of the BstU I reaction, which was incubated at 60 °C. .. The restriction fragments were desalted using Sephadex G-75 (Amersham Biosciences, Uppsala, Sweden) and dehydrated.

    Article Title: An association study of HFE gene mutation with idiopathic male infertility in the Chinese Han population
    Article Snippet: .. The PCR products were then digested at 37 °C for 16 h with 5 U of Mbo I, 2.5 U of Hinf I or 2.5 U of Rsa I in a 10-µl reaction mixture ( New England Biolabs Co., Ltd., Beijing, China; ). .. Finally, the restriction fragments were separated by electrophoresis on 4% agarose gels, and the results were visualised under UV illumination (JS-680B; Shanghai Peiqing Science & Technology.

    Article Title: A System for the Continuous Directed Evolution of Biomolecules
    Article Snippet: .. Enzymes were denatured at 85°C for 5 minutes followed by DNA amplification with sequential PCR reactions using primers 5’-CGATCCGAACGCAGCATTTACGCTGATGGCGATGAATGAACACTG-3’ and 5’-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3’, digestion with Mly I and Hinf I in NEBuffer 4 to cleave sequences containing the promoter, and PCR amplification with 5’-GCTAGTTATTGCTCAGCGGAATAACGATCCGAACGCAGCATTTAC-3’ and 5’-GCTAGTTATTGCTCAGCGGAAAAAAAAAAAAACCCCCAAACCCCC-3’. .. Enzymes were denatured at 85°C for 5 minutes followed by DNA amplification with sequential PCR reactions using primers 5’-CGATCCGAACGCAGCATTTACGCTGATGGCGATGAATGAACACTG-3’ and 5’-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3’, digestion with Mly I and Hinf I in NEBuffer 4 to cleave sequences containing the promoter, and PCR amplification with 5’-GCTAGTTATTGCTCAGCGGAATAACGATCCGAACGCAGCATTTAC-3’ and 5’-GCTAGTTATTGCTCAGCGGAAAAAAAAAAAAACCCCCAAACCCCC-3’.

    Article Title: Investigation of associations of ARMS2, CD14, and TLR4 gene polymorphisms with wet age-related macular degeneration in a Greek population
    Article Snippet: With regard to the TLR4 amplicons after digestion with the Nco I (Takara, Japan) restriction enzyme for the 299 residue and with Hinf I (NEB, USA) for the 399 residue, fragment sizes for carriers of the polymorphic allele decreased from 249 (wild-type) to 223 bp for the 299 residue and from 406 (wild-type) to 377 bp for the 399 residue ( and ). .. All restricted amplicons were analyzed by 3% agarose gel electrophoresis (2:1 Nusieve/Seakem, Lonza, Basel, Switzerland), visualized by ethidium bromide and sized by a MW marker (PCR Marker or ϕχ174 Hae III, both New England Biolabs, Ipswich, MA, USA).

    Article Title: Epidemiological and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from the Ear Discharge of Outpatients with Chronic Otitis Media
    Article Snippet: .. PCR products were purified by the QIAquick-spin PCR purification kit (Qiagen Inc., Valencia, CA, U.S.A.) and followed by digestion with Hinf I and Bsm I (New England Biolabs Inc., Ipswich, MA, U.S.A.). .. The RFLP patterns were analyzed using 2% agarose gel electrophoresis.

    Article Title: The Association of Methylenetetrahydrofolate Reductase Genotypes with the Risk of Childhood Leukemia in Taiwan
    Article Snippet: .. The 198-bp PCR product of MTHFR C677T and 138-bp PCR product of MTHFR A1298C were subject to enzyme digestion with Hinf I and Fnu4H I (New England, Biolabs, Beverly, MA USA), respectively for 4 h and then visualized by ethidium bromide-stained 3% agarose gel electrophoresis under UV light. .. On digestion with Hinf I , the PCR product of MTHFR C677T arising from the C allele was uncut (198 bp), whereas the T allele was cut into fragments of 175 bp and 23 bp.

    Article Title: Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease
    Article Snippet: .. After amplification, 8 μl of the PCR products were mixed and digested with 1μl (10000U/ml) Hinf I (New England BioLabs, Ipswich, USA) and 1μl 10 x buffer and then incubated for 3 hours at 37°C. ..

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: .. Identification of a full length Aphenenchoides besseyi GH5 gene by inverse and RACE PCR Nematode genomic DNA (~50 ng) was digested with Hinf I (NEB, California, USA, #Ro155S) and self-ligated with T4 DNA Ligase (GeneMark, Taipei, Taiwan) according to the manufacturer’s protocol. .. The two inverse primers FmGH5i-F and FmGH5i-R ( ) were designed and synthesized on the basis of the sequence for genomic DNA fragments obtained with the degenerate primers through the LASERGENE® PrimerSelect™ program, and the PCR reaction was started with a 3 min denaturation at 94°C, followed by 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min 30 s, and a final extension at 72°C for 5 min.

    Blocking Assay:

    Article Title: Transient introduction of human telomerase mRNA improves hallmarks of progeria cells, et al. Transient introduction of human telomerase mRNA improves hallmarks of progeria cells
    Article Snippet: DNA (3 μg) was digested with HinF I and Rsa I (NEB) overnight and separated on a 0.85% agarose gel at 2 V/cm for 16 hr. .. The membrane was incubated with 1× DIG blocking solution and then with anti‐DIG antibody (Roche) for 30 min. Telomere signals were detected by incubating with CDP‐star (Roche) for 5 min.

    Activated Clotting Time Assay:

    Article Title: Epidemiological and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from the Ear Discharge of Outpatients with Chronic Otitis Media
    Article Snippet: The 643-bp ccrB gene fragment was amplified using a pair of primers: CRB-1 (5'-GGC TAT TAT CAA GGC AAT TTA CC -3'), and CRB-2 (5'-ACT TTA TCA CTT TTG ACT ATT TCG 3'). .. PCR products were purified by the QIAquick-spin PCR purification kit (Qiagen Inc., Valencia, CA, U.S.A.) and followed by digestion with Hinf I and Bsm I (New England Biolabs Inc., Ipswich, MA, U.S.A.).

    In Situ Hybridization:

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: .. Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C. .. The digested DNA was separated on a 0.8% agarose gel and blotted onto a positively charged nylon membrane (GE Healthcare Biosciences).

    Plasmid Preparation:

    Article Title: A System for the Continuous Directed Evolution of Biomolecules
    Article Snippet: Enzymes were denatured at 85°C for 5 minutes followed by DNA amplification with sequential PCR reactions using primers 5’-CGATCCGAACGCAGCATTTACGCTGATGGCGATGAATGAACACTG-3’ and 5’-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3’, digestion with Mly I and Hinf I in NEBuffer 4 to cleave sequences containing the promoter, and PCR amplification with 5’-GCTAGTTATTGCTCAGCGGAATAACGATCCGAACGCAGCATTTAC-3’ and 5’-GCTAGTTATTGCTCAGCGGAAAAAAAAAAAAACCCCCAAACCCCC-3’. .. Enzymes were denatured at 85°C for 5 minutes followed by DNA amplification with sequential PCR reactions using primers 5’-CGATCCGAACGCAGCATTTACGCTGATGGCGATGAATGAACACTG-3’ and 5’-CCCCCAAACCCCCAAAAAAAAAACCCACCCAAAAAAAAAAAA-3’, digestion with Mly I and Hinf I in NEBuffer 4 to cleave sequences containing the promoter, and PCR amplification with 5’-GCTAGTTATTGCTCAGCGGAATAACGATCCGAACGCAGCATTTAC-3’ and 5’-GCTAGTTATTGCTCAGCGGAAAAAAAAAAAAACCCCCAAACCCCC-3’.

    Electrophoresis:

    Article Title: Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii
    Article Snippet: PCR products (15 μl) were partitioned into eight aliquots and separately digested overnight with 5 U of Hae III, Hha I, Alu I, Mbo I, Msp I, Rsa I, Hinf I and BstU I (New England Biolabs, Ipswich, MA, USA) in a total volume of 30 μl at 37 °C, with the exception of the BstU I reaction, which was incubated at 60 °C. .. Fragments were resuspended in 15 μl formamide and 0.33 μl Genescan ROX-500 internal size standard (Applied Biosystems, Foster City, CA, USA), denatured by heating for 5 min at 95 °C, and separated by capillary electrophoresis using an ABI 3100 genetic analyzer with a 50 cm capillary array using POP6 polymer (Applied Biosystems).

    Article Title: An association study of HFE gene mutation with idiopathic male infertility in the Chinese Han population
    Article Snippet: The PCR products were then digested at 37 °C for 16 h with 5 U of Mbo I, 2.5 U of Hinf I or 2.5 U of Rsa I in a 10-µl reaction mixture ( New England Biolabs Co., Ltd., Beijing, China; ). .. Finally, the restriction fragments were separated by electrophoresis on 4% agarose gels, and the results were visualised under UV illumination (JS-680B; Shanghai Peiqing Science & Technology.

    Article Title: Senescent stroma promotes prostate cancer progression: The role of miR‐210), Senescent stroma promotes prostate cancer progression: The role of miR‐210
    Article Snippet: .. Five micrograms of DNA were digested with Hinf I (10 U/μl, New England Biolabs, Ipswich, MA) and resolved on a 1.2% agarose gel by pulsed field gel electrophoresis (5 V/cm, 0.5–5 s switch times, 14 °C for 17 h). .. Lambda DNA‐Hind III digest (Bio‐Rad Laboratories, Segrate, Italy) was used for size determination of telomere restriction fragments (TRF).

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride
    Article Snippet: Genomic DNA was digested with an Rsa I and Hinf I (NEB) endonuclease mixture (1:1) at 37 °C overnight and then resolved in a 1% agarose gel. .. After electrophoresis, the gel was stained with FluoroStain™ DNA Fluorescent Staining Dye (SMOBIO) to check the quality of DNA digestion.

    Agarose Gel Electrophoresis:

    Article Title: Ultra-diffuse hydrothermal venting supports Fe-oxidizing bacteria and massive umber deposition at 5000 m off Hawaii
    Article Snippet: PCR products were visually assayed for size by 1% agarose gel electrophoresis against a 1-kb ladder DNA size standard. .. PCR products (15 μl) were partitioned into eight aliquots and separately digested overnight with 5 U of Hae III, Hha I, Alu I, Mbo I, Msp I, Rsa I, Hinf I and BstU I (New England Biolabs, Ipswich, MA, USA) in a total volume of 30 μl at 37 °C, with the exception of the BstU I reaction, which was incubated at 60 °C.

    Article Title: Investigation of associations of ARMS2, CD14, and TLR4 gene polymorphisms with wet age-related macular degeneration in a Greek population
    Article Snippet: With regard to the TLR4 amplicons after digestion with the Nco I (Takara, Japan) restriction enzyme for the 299 residue and with Hinf I (NEB, USA) for the 399 residue, fragment sizes for carriers of the polymorphic allele decreased from 249 (wild-type) to 223 bp for the 299 residue and from 406 (wild-type) to 377 bp for the 399 residue ( and ). .. All restricted amplicons were analyzed by 3% agarose gel electrophoresis (2:1 Nusieve/Seakem, Lonza, Basel, Switzerland), visualized by ethidium bromide and sized by a MW marker (PCR Marker or ϕχ174 Hae III, both New England Biolabs, Ipswich, MA, USA).

    Article Title: Epidemiological and Genetic Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from the Ear Discharge of Outpatients with Chronic Otitis Media
    Article Snippet: PCR products were purified by the QIAquick-spin PCR purification kit (Qiagen Inc., Valencia, CA, U.S.A.) and followed by digestion with Hinf I and Bsm I (New England Biolabs Inc., Ipswich, MA, U.S.A.). .. The RFLP patterns were analyzed using 2% agarose gel electrophoresis.

    Article Title: Senescent stroma promotes prostate cancer progression: The role of miR‐210), Senescent stroma promotes prostate cancer progression: The role of miR‐210
    Article Snippet: .. Five micrograms of DNA were digested with Hinf I (10 U/μl, New England Biolabs, Ipswich, MA) and resolved on a 1.2% agarose gel by pulsed field gel electrophoresis (5 V/cm, 0.5–5 s switch times, 14 °C for 17 h). .. Lambda DNA‐Hind III digest (Bio‐Rad Laboratories, Segrate, Italy) was used for size determination of telomere restriction fragments (TRF).

    Article Title: The Association of Methylenetetrahydrofolate Reductase Genotypes with the Risk of Childhood Leukemia in Taiwan
    Article Snippet: .. The 198-bp PCR product of MTHFR C677T and 138-bp PCR product of MTHFR A1298C were subject to enzyme digestion with Hinf I and Fnu4H I (New England, Biolabs, Beverly, MA USA), respectively for 4 h and then visualized by ethidium bromide-stained 3% agarose gel electrophoresis under UV light. .. On digestion with Hinf I , the PCR product of MTHFR C677T arising from the C allele was uncut (198 bp), whereas the T allele was cut into fragments of 175 bp and 23 bp.

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C. .. The digested DNA was separated on a 0.8% agarose gel and blotted onto a positively charged nylon membrane (GE Healthcare Biosciences).

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride
    Article Snippet: .. Genomic DNA was digested with an Rsa I and Hinf I (NEB) endonuclease mixture (1:1) at 37 °C overnight and then resolved in a 1% agarose gel. .. After electrophoresis, the gel was stained with FluoroStain™ DNA Fluorescent Staining Dye (SMOBIO) to check the quality of DNA digestion.

    Article Title: Transient introduction of human telomerase mRNA improves hallmarks of progeria cells, et al. Transient introduction of human telomerase mRNA improves hallmarks of progeria cells
    Article Snippet: .. DNA (3 μg) was digested with HinF I and Rsa I (NEB) overnight and separated on a 0.85% agarose gel at 2 V/cm for 16 hr. ..

    Ethanol Precipitation:

    Article Title: FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops
    Article Snippet: Genomic DNA analysis Genomic DNA was isolated by phenol:chloroform extraction and treatment with 40 μg/ml RNaseA, followed by ethanol precipitation. .. Reconstituted DNA was digested with Hinf I and Rsa I (New England Biolabs) and again purified by phenol:chloroform extraction.

    Molecular Weight:

    Article Title: Global Methylation Profiling of Lung Cancer Identifies Novel Methylated Genes 1
    Article Snippet: In summary, high molecular weight DNA was digested with the methylation sensitive restriction enzyme Not I (Promega, Madison, WI), end-labeled by [ α -32 P]dGTP and [ α -32 P]dCTP (Amersham, Piscataway, NJ), and then digested using the restriction enzyme Eco RV (Promega). .. Not I- Eco RV DNA fragments were separated in a first dimension through a 0.8% agarose tube gel, followed by an in-gel digestion with a third restriction enzyme, Hinf I (New England Biolabs, Beverly MA).

    CTG Assay:

    Article Title: The Association of Methylenetetrahydrofolate Reductase Genotypes with the Risk of Childhood Leukemia in Taiwan
    Article Snippet: The primers for MTHFR C677T were forward 5’- TGA AGG AGA AGG TGT CTG CGG GA-3’ and reverse 5’- AGG ACG GTG CGG TGA GAG TG-3’. .. The 198-bp PCR product of MTHFR C677T and 138-bp PCR product of MTHFR A1298C were subject to enzyme digestion with Hinf I and Fnu4H I (New England, Biolabs, Beverly, MA USA), respectively for 4 h and then visualized by ethidium bromide-stained 3% agarose gel electrophoresis under UV light.

    Staining:

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride
    Article Snippet: Genomic DNA was digested with an Rsa I and Hinf I (NEB) endonuclease mixture (1:1) at 37 °C overnight and then resolved in a 1% agarose gel. .. After electrophoresis, the gel was stained with FluoroStain™ DNA Fluorescent Staining Dye (SMOBIO) to check the quality of DNA digestion.

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    New England Biolabs hinfi restriction enzymes
    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a <t>HinfI</t> restriction site when C2268 is methylated. Below: Cleavage of <t>PCR</t> amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product
    Hinfi Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Journal: BMC Plant Biology

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

    doi: 10.1186/s12870-015-0580-8

    Figure Lengend Snippet: NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Article Snippet: PCR products were digested with HpyCH4IV or HinfI restriction enzymes (New England Biolabs), respectively.

    Techniques: Methylation, Amplification, Polymerase Chain Reaction

    Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Journal: PLoS ONE

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    doi: 10.1371/journal.pone.0158663

    Figure Lengend Snippet: Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Article Snippet: Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C.

    Techniques: Southern Blot, Isolation

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    doi: 10.1093/nar/gky597

    Figure Lengend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Article Snippet: Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel.

    Techniques: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    doi: 10.1093/nar/gky597

    Figure Lengend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Article Snippet: Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization