hin diii New England Biolabs Search Results


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  • 99
    New England Biolabs hin diii
    Southern blots of Hin <t>dIII</t> and Hin dIII-VDE digests of <t>DNA</t> from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015
    Hin Diii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3091 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs hin diii hf
    Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin <t>dIII</t> restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key <t>DNA</t> fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)
    Hin Diii Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs hin diii restriction endonuclease
    PCV3 viral gene rearrangement. (A) Rearranged linear gene sequence of PCV3 containing Hin <t>dIII</t> restriction sites. The Hin dIII restriction site acted as the reopening site, added to the end of the linear sequence after the opening process. (B) <t>PCR</t> detection after PCV3 cyclization. Specific primers, covering PCV3 M2 and PCV3 L3, were designed to verify whether the PCV3 gene sequence was cyclized. However, the PCV3 linear sequence could not be amplified by the mentioned primers.
    Hin Diii Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna ligase
    Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by <t>T4</t> DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs lambda dna hin diii digest
    Effect of reservoir buffer solution on MRT preconcentration. A schematic describing the experimental preconcentration conditions is shown on the left, and the single-molecule chromatogram after separation is shown on the right. In all conditions, 2× EB + 18 mM HCl was used as the running buffer. a Control separation of <t>λ</t> DNA Hin <t>dIII</t> digest without preconcentration or a buffer mismatch: the reservoir buffer is the same as the running buffer and no counterflow is applied. b Counterflow at 100 psi for 12 min into reservoir buffer containing Hin dIII digested λ DNA in DI water. The DNA, originally suspended at 0.005 ng/μL concentrates 1000-fold prior to separation. c A similar 1000-fold concentration factor is achieved with the reservoir buffer solutions containing DNA prepared in 2× EB by flowing at a 4-fold higher flow rate (400 psi for 12 min)
    Lambda Dna Hin Diii Digest, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs pmal c5x vector
    Effect of reservoir buffer solution on MRT preconcentration. A schematic describing the experimental preconcentration conditions is shown on the left, and the single-molecule chromatogram after separation is shown on the right. In all conditions, 2× EB + 18 mM HCl was used as the running buffer. a Control separation of <t>λ</t> DNA Hin <t>dIII</t> digest without preconcentration or a buffer mismatch: the reservoir buffer is the same as the running buffer and no counterflow is applied. b Counterflow at 100 psi for 12 min into reservoir buffer containing Hin dIII digested λ DNA in DI water. The DNA, originally suspended at 0.005 ng/μL concentrates 1000-fold prior to separation. c A similar 1000-fold concentration factor is achieved with the reservoir buffer solutions containing DNA prepared in 2× EB by flowing at a 4-fold higher flow rate (400 psi for 12 min)
    Pmal C5x Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 712 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xba i
    Effect of reservoir buffer solution on MRT preconcentration. A schematic describing the experimental preconcentration conditions is shown on the left, and the single-molecule chromatogram after separation is shown on the right. In all conditions, 2× EB + 18 mM HCl was used as the running buffer. a Control separation of <t>λ</t> DNA Hin <t>dIII</t> digest without preconcentration or a buffer mismatch: the reservoir buffer is the same as the running buffer and no counterflow is applied. b Counterflow at 100 psi for 12 min into reservoir buffer containing Hin dIII digested λ DNA in DI water. The DNA, originally suspended at 0.005 ng/μL concentrates 1000-fold prior to separation. c A similar 1000-fold concentration factor is achieved with the reservoir buffer solutions containing DNA prepared in 2× EB by flowing at a 4-fold higher flow rate (400 psi for 12 min)
    Xba I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs bss hii
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Bss Hii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    New England Biolabs bgl i
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Bgl I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs calf intestinal alkaline phosphatase
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Calf Intestinal Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2886 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs klenow fragment
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7893 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs gibson assembly
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Gibson Assembly, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega hin diii restriction enzymes
    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, <t>Bss</t> <t>HII-digested</t> 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.
    Hin Diii Restriction Enzymes, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques: Mutagenesis

    70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Journal: Human genetics

    Article Title: 3C-PCR: A novel proximity ligation-based approach to phase chromosomal rearrangement breakpoints with distal allelic variants

    doi: 10.1007/s00439-017-1853-0

    Figure Lengend Snippet: Assay validation. a The goal of the assay in the DGAP230 experimental system is to differentiate the target region (yellow box) on the der(20) chromosome (top) from the target region on the normal chr20 (bottom). The small green bar represents the 3C genomic fragment that contains the target region and the small blue bar represents the digested genomic fragment containing a breakpoint-proximal region from the segment of chr22 translocated to the der(20). Rough gray edges reflect enzymatic digestion at flanking Hin dIII restriction sites. b Schematic of nested PCR amplifications for the predicted ligation product with the target region (green bar above mahogany map) and the chr22 fragment (blue bar above light pink map). c Gel electrophoresis displays products from the first PCR across the breakpoint for experimental and control 3C libraries (left), and the second nested PCR (right, N=3). Key DNA fragment sizes of the markers (M) are indicated on the left. d Sanger sequencing traces of the target variable region from the nested PCR amplicon (top) and genomic DNA from the same cell line (bottom; N=3)

    Article Snippet: Chromatin was digested with Hin dIII-HF (NEB), ligated with T4 DNA ligase (NEB), and reverse crosslinked by incubation with Proteinase K (NEB) and RNase A (EMD Millipore).

    Techniques: Nested PCR, Ligation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Sequencing, Amplification

    PCV3 viral gene rearrangement. (A) Rearranged linear gene sequence of PCV3 containing Hin dIII restriction sites. The Hin dIII restriction site acted as the reopening site, added to the end of the linear sequence after the opening process. (B) PCR detection after PCV3 cyclization. Specific primers, covering PCV3 M2 and PCV3 L3, were designed to verify whether the PCV3 gene sequence was cyclized. However, the PCV3 linear sequence could not be amplified by the mentioned primers.

    Journal: Frontiers in Microbiology

    Article Title: A Novel Technique for Constructing Infectious Cloning of Type 3 Porcine Circovirus

    doi: 10.3389/fmicb.2020.01067

    Figure Lengend Snippet: PCV3 viral gene rearrangement. (A) Rearranged linear gene sequence of PCV3 containing Hin dIII restriction sites. The Hin dIII restriction site acted as the reopening site, added to the end of the linear sequence after the opening process. (B) PCR detection after PCV3 cyclization. Specific primers, covering PCV3 M2 and PCV3 L3, were designed to verify whether the PCV3 gene sequence was cyclized. However, the PCV3 linear sequence could not be amplified by the mentioned primers.

    Article Snippet: Subsequently, the purified PCR product was digested by Hin dIII restriction endonuclease (NEB, Beijing, China) and then incubated in a water bath at 37°C for 5 h. Then, the digested products of HindIII restriction endonuclease were purified following the manufacturer’s instructions (Takara Bio, Dalian, China).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification

    Electrophoresis of recombinant plasmid DNA. Agarose gel (0.8%) showing the results of PCR and the digested clone fragments. Lanes: M1, λDNA/ Hin dIII DNA marker; 1, digested clone fragments of pET32c-Ang-2 plasmid; 2, Ang-2 gene; M2, DL2000 DNA marker. Ang-2, angiopoietin-2.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Suppression of angiogenesis and tumor growth in vitro and in vivo using an anti-angiopoietin-2 single-chain antibody

    doi: 10.3892/etm.2014.1476

    Figure Lengend Snippet: Electrophoresis of recombinant plasmid DNA. Agarose gel (0.8%) showing the results of PCR and the digested clone fragments. Lanes: M1, λDNA/ Hin dIII DNA marker; 1, digested clone fragments of pET32c-Ang-2 plasmid; 2, Ang-2 gene; M2, DL2000 DNA marker. Ang-2, angiopoietin-2.

    Article Snippet: Reagents The following reagents were obtained: pET32c vector system from Novogen (Madison, WI, USA); plasmid pCANTAB5E, Escherichia coli TG1 and Escherichia coli BL21, M13K07 helper phage, mouse anti-M13 antibody and mouse anti-E tag antibody from Pharmacia Biotech (Piscataway, NJ, USA); pfuDNA polymerase from Stratagene (Santa Clara, CA, USA), restriction endonuclease Hin dIII, Nco I and T4 DNA Ligase from New England Biolabs (Ipswich, MA, USA); low melting point agarose from Promega (Madison, WI, USA); isopropyl β-D-1-thiogalactopyranoside (IPTG), total RNA kit and Moloney murine leukemia virus (M-MLV) reverse transcriptase from Takara (Shiga, Japan); protein marker and FastDigest restriction enzymes from MBI Fermentas Inc. (Burlington, ON, Canada); Rapid Gel purification kit and PureLink™ HiPure plasmid DNA purification kit from Invitrogen (Carlsbad, CA, USA); enterokinase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma (St. Louis, MO, USA); M199, fetal bovine serum (FBS) and trypsin-ethylenediaminetetraacetic acid (EDTA) from Hyclone (Logan, UT, USA); Dulbecco’s modified Eagle’s medium (DMEM) from PAA Laboratories GmbH (Linz, Austria); endothelial cell growth supplement ECGs) from Millipore (Temecula, CA, USA); collagenase from Worthington Biochemica (Lakewood, NJ, USA); mouse anti-human CD31 monoclonal antibody from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA); EDTA, penicillin and streptomycin from Invitrogen; and dimethyl sulfoxide (DMSO) and Transwell chambers with 8-μm pore filters from Corning Incorporated (Corning, NY, USA).

    Techniques: Electrophoresis, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Marker

    Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.

    Journal: PLoS Biology

    Article Title: The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans

    doi: 10.1371/journal.pbio.2001164

    Figure Lengend Snippet: Mlh1-Mlh3 can make DSBs on large DNA substrates. (A) Mlh1-Mlh3 makes exclusively linear product from a closed circular substrate approximately 12 kb in size. Experiments were performed identical to the manner described in Fig 2C using the indicated sized plasmids. Red asterisk indicates location where nicked 12 kb plasmid migrates. (B) 15 μM total nucleotide 12 kb circular substrate was incubated with 300 nM Mlh1-Mlh3 for the indicated period of time. Plasmid linearized with Hin dIII was used as a marker for linear product in the lane 8 of the left panel. 12 kb plasmid treated with DNaseI is used as a marker for closed circular, linear, and nicked species in lane 1. Lane 9 is a negative control reaction in which Mlh1-Mlh3 was omitted to indicate migration of closed circular substrate. The plot indicates quantification of the representative gel shown. (C) Experiment is identical to that conducted in B, except 15 μM total nucleotide 2.7 kb circular substrate was used. The plot indicates quantification of the representative gel shown. (D) Native gel analysis of material in Fig 2E lanes 7–11. (E). DSBs made by Mlh1-Mlh3 can be religated. 12 kb linear product from Mlh1-Mlh3 endonuclease assay (“Mlh1-Mlh3 linear pdt”) was gel isolated and incubated with T4 polymerase where indicated with a + (lanes 12–13) followed by T4 DNA ligase where indicated with a + (lanes 11, 13). As controls, 12 kb closed circular plasmid was linearized with either Sca I or Hin dIII and religated (lanes 4–7) or linearized with Hin dIII and blunted with T4 polymerase followed by a religation step (lanes 8–9). Gel-isolated 12 kb closed circular DNA and Sca I-linearized DNA were ran in lanes 2–3 as migration markers.

    Article Snippet: Relaxed circular pUC18 plasmid was prepared by linearization with Hin dIII, followed by treatment with T4 DNA ligase (NEB).

    Techniques: Plasmid Preparation, Incubation, Marker, Negative Control, Migration, Isolation

    Effect of reservoir buffer solution on MRT preconcentration. A schematic describing the experimental preconcentration conditions is shown on the left, and the single-molecule chromatogram after separation is shown on the right. In all conditions, 2× EB + 18 mM HCl was used as the running buffer. a Control separation of λ DNA Hin dIII digest without preconcentration or a buffer mismatch: the reservoir buffer is the same as the running buffer and no counterflow is applied. b Counterflow at 100 psi for 12 min into reservoir buffer containing Hin dIII digested λ DNA in DI water. The DNA, originally suspended at 0.005 ng/μL concentrates 1000-fold prior to separation. c A similar 1000-fold concentration factor is achieved with the reservoir buffer solutions containing DNA prepared in 2× EB by flowing at a 4-fold higher flow rate (400 psi for 12 min)

    Journal: Nature Communications

    Article Title: Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow

    doi: 10.1038/s41467-017-01214-y

    Figure Lengend Snippet: Effect of reservoir buffer solution on MRT preconcentration. A schematic describing the experimental preconcentration conditions is shown on the left, and the single-molecule chromatogram after separation is shown on the right. In all conditions, 2× EB + 18 mM HCl was used as the running buffer. a Control separation of λ DNA Hin dIII digest without preconcentration or a buffer mismatch: the reservoir buffer is the same as the running buffer and no counterflow is applied. b Counterflow at 100 psi for 12 min into reservoir buffer containing Hin dIII digested λ DNA in DI water. The DNA, originally suspended at 0.005 ng/μL concentrates 1000-fold prior to separation. c A similar 1000-fold concentration factor is achieved with the reservoir buffer solutions containing DNA prepared in 2× EB by flowing at a 4-fold higher flow rate (400 psi for 12 min)

    Article Snippet: Sample preparation For fluorescent microscope experiments, Hin dIII digested λ DNA (New England Biolabs, Inc.) was diluted to 10 ng/μL and stained with 40× or 100× dilution of PicoGreen (Thermo Fisher Scientific, Inc.) in 1 mM HEPES-Tris buffer.

    Techniques: Concentration Assay, Flow Cytometry

    Coupled preconcentration, free solution hydrodynamic separation, and single-molecule detection platform. Each end of a long microcapillary is placed into its own individually controled pressure chamber to enable precise control over the direction and speed of fluid flow. Concentration occurs by applying positive pressure to the running buffer pressure chamber with Pressure Source 1, and sample injection and separation occur with positive pressure applied to the opposite end of the capillary with Pressure Source 2. The CICS observation volume is aligned with the detection region to enable detection of single DNA molecules (grey, background), which are counted to generate a single-molecule chromatogram (navy, shaded foreground). This example chromatogram was generated after performing 100× MRT preconcentration and free solution separation of Hin dIII digested λ DNA

    Journal: Nature Communications

    Article Title: Molecular rheotaxis directs DNA migration and concentration against a pressure-driven flow

    doi: 10.1038/s41467-017-01214-y

    Figure Lengend Snippet: Coupled preconcentration, free solution hydrodynamic separation, and single-molecule detection platform. Each end of a long microcapillary is placed into its own individually controled pressure chamber to enable precise control over the direction and speed of fluid flow. Concentration occurs by applying positive pressure to the running buffer pressure chamber with Pressure Source 1, and sample injection and separation occur with positive pressure applied to the opposite end of the capillary with Pressure Source 2. The CICS observation volume is aligned with the detection region to enable detection of single DNA molecules (grey, background), which are counted to generate a single-molecule chromatogram (navy, shaded foreground). This example chromatogram was generated after performing 100× MRT preconcentration and free solution separation of Hin dIII digested λ DNA

    Article Snippet: Sample preparation For fluorescent microscope experiments, Hin dIII digested λ DNA (New England Biolabs, Inc.) was diluted to 10 ng/μL and stained with 40× or 100× dilution of PicoGreen (Thermo Fisher Scientific, Inc.) in 1 mM HEPES-Tris buffer.

    Techniques: Flow Cytometry, Concentration Assay, Injection, Generated

    (A) Agarose gel electrophoresis of Eco RI-digested genomic and plasmid DNAs from VREF isolates. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain; 3, chromosomal DNA from a known VanB strain; 4 and 5, genomic and plasmid DNA from a group 4 isolate ( vanA gene on chromosome); 6 and 7, genomic and plasmid DNA from a group 4 isolate ( vanA gene on plasmid); 8 and 9, genomic and plasmid DNA from a group 11 isolate ( vanA gene on plasmid); 10 to 12, genomic DNAs from VanB isolates (group 23, group 8, and group 9, respectively). (B) Southern blot of gel shown in panel A probed with biotin-labeled vanA gene. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain positive for the vanA gene; 3, chromosomal DNA from a known VanB strain negative for the vanA gene; 4, genomic DNA from an isolate positive for the vanA gene; 5, plasmid DNA from isolate in lane 4 negative for the vanA gene; 6, genomic DNA from isolate positive for the vanA gene; 7, plasmid DNA from the isolate in lane 6 also positive for the vanA gene; 8, genomic DNA from isolate positive for the vanA gene; 9, plasmid DNA from the isolate in lane 8 positive for the vanA gene; 10 to 12, genomic DNAs from isolates negative for the vanA gene. (C) Southern blot of gel in shown in panel A probed with biotin-labeled vanB gene. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain negative for the vanB gene; 3, chromosomal DNA from a known VanB strain positive for the vanB gene; 4 to 9, genomic and plasmid DNAs from these isolates negative for the vanB gene; 10 to 12, genomic DNAs from these isolates positive for the vanB gene.

    Journal: Journal of Clinical Microbiology

    Article Title: Molecular Analysis of Glycopeptide-Resistant Enterococcus faecium Isolates Collected from Michigan Hospitals over a 6-Year Period

    doi:

    Figure Lengend Snippet: (A) Agarose gel electrophoresis of Eco RI-digested genomic and plasmid DNAs from VREF isolates. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain; 3, chromosomal DNA from a known VanB strain; 4 and 5, genomic and plasmid DNA from a group 4 isolate ( vanA gene on chromosome); 6 and 7, genomic and plasmid DNA from a group 4 isolate ( vanA gene on plasmid); 8 and 9, genomic and plasmid DNA from a group 11 isolate ( vanA gene on plasmid); 10 to 12, genomic DNAs from VanB isolates (group 23, group 8, and group 9, respectively). (B) Southern blot of gel shown in panel A probed with biotin-labeled vanA gene. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain positive for the vanA gene; 3, chromosomal DNA from a known VanB strain negative for the vanA gene; 4, genomic DNA from an isolate positive for the vanA gene; 5, plasmid DNA from isolate in lane 4 negative for the vanA gene; 6, genomic DNA from isolate positive for the vanA gene; 7, plasmid DNA from the isolate in lane 6 also positive for the vanA gene; 8, genomic DNA from isolate positive for the vanA gene; 9, plasmid DNA from the isolate in lane 8 positive for the vanA gene; 10 to 12, genomic DNAs from isolates negative for the vanA gene. (C) Southern blot of gel in shown in panel A probed with biotin-labeled vanB gene. Lanes: 1, biotin-labeled Hin dIII-digested lambda phage DNA; 2, chromosomal DNA from a known VanA strain negative for the vanB gene; 3, chromosomal DNA from a known VanB strain positive for the vanB gene; 4 to 9, genomic and plasmid DNAs from these isolates negative for the vanB gene; 10 to 12, genomic DNAs from these isolates positive for the vanB gene.

    Article Snippet: Biotinylated lambda DNA/ Hin dIII fragments were used as size markers (New England BioLabs).

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Labeling, Southern Blot

    Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, Bss HII-digested 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.

    Journal: Journal of Virology

    Article Title: Murine Cytomegalovirus CC Chemokine Homolog MCK-2 (m131-129) Is a Determinant of Dissemination That Increases Inflammation at Initial Sites of Infection

    doi: 10.1128/JVI.75.20.9966-9976.2001

    Figure Lengend Snippet: Restriction digestion analysis of RM4511 DNA. (A) Detection of the RM4511 mck mutation by Bbr PI digestion. Autoradiograph of Bbr PI-digested (Roche) virion DNA from parental RM427 + , RM4503, and two isolates of RM4511 (RM4511.1 and RM4511.2) following electrophoretic separation on a 1% agarose gel and hybridization with a Hin dIII/ Afl II fragment mck probe. (B) Detection of the EGFP-puro insert in RM4503 and RM4511. Autoradiograph of electrophoretically separated, Bss HII-digested 32 P-end-labeled DNA fragments. (C) Autoradiograph of electrophoretically separated 32 P-end-labeled Hin dIII fragments of DNA from RM427 + , RM4503, and two isolates of RM4511.

    Article Snippet: Virion DNA (0.5 to 1.0 μg) was digested with Hin dIII, Bss HII, Afl II, or Spe I (New England Biolabs, Beverly, Mass.) and end labeled in the presence of 2.5 μCi of [α-32 P]dCTP (Amersham), 125 μM each dATP, dGTP, and dTTP, and 0.5 U of Klenow polymerase (Roche, Indianapolis, Ind.) for 15 min at room temperature in 20 μl of restriction enzyme buffer.

    Techniques: Mutagenesis, Autoradiography, Agarose Gel Electrophoresis, Hybridization, Labeling