apali  (New England Biolabs)


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    Name:
    ApaLI
    Description:
    ApaLI 12 500 units
    Catalog Number:
    r0507l
    Price:
    269
    Size:
    12 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs apali
    ApaLI
    ApaLI 12 500 units
    https://www.bioz.com/result/apali/product/New England Biolabs
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    apali - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein"

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.780239

    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
    Figure Legend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Techniques Used: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    2) Product Images from "Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease"

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp643

    Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).
    Figure Legend Snippet: Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Techniques Used: Southern Blot, Clone Assay, Transfection, Derivative Assay, Plasmid Preparation, Molecular Weight, Amplification, Binding Assay, Marker

    PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.
    Figure Legend Snippet: PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Techniques Used: Polymerase Chain Reaction, Genetically Modified, Stable Transfection, Nested PCR, Clone Assay, Derivative Assay, Transfection, Plasmid Preparation, Labeling, Amplification, Negative Control, Marker, Molecular Weight, Agarose Gel Electrophoresis

    3) Product Images from "Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells"

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2804

    Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.
    Figure Legend Snippet: Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Techniques Used: Activation Assay, Western Blot, Incubation, Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    4) Product Images from "RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation"

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp780

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Figure Legend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Techniques Used: Southern Blot, Staining, Produced, Generated

    Related Articles

    Clone Assay:

    Article Title: Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule *
    Article Snippet: Paragraph title: wnk Cloning ... The resultant product, as well as pENTR containing segment one, was then digested with ApaLI (New England Biolabs, Ipswich, MA) and ligated together to yield the final full-length wnk cDNA in pENTR.

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: The resulting plasmid contains CEN4 cloned in between S. cerevisiae URA3 and a hygromycin-resistance expression cassette ( HygR ). .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG.

    Amplification:

    Article Title: Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule *
    Article Snippet: The third segment was amplified with primers W13711D and W15754U. .. The resultant product, as well as pENTR containing segment one, was then digested with ApaLI (New England Biolabs, Ipswich, MA) and ligated together to yield the final full-length wnk cDNA in pENTR.

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer. .. The 393-bp AAVS1 -specific probe was obtained by PCR amplification of human chromosomal DNA using 0.012 U/µl Phusion High-Fidelity DNA polymerase (Finnzymes), 200 µM deoxynucleoside triphosphates (dNTPs; Fermentas), 1× GC buffer (Finnzymes) plus 0.2 µM of primers #633 (5′-GGTCCCCAGCATGTCTTCCTA-3′) and #634 (5′-CTCCCGAACCTCAGATCTCC-3′).

    Construct:

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: To convert a linear YAC into a cYAC or to assemble DNA fragments with overlapping ends into a single cYAC in S. cerevisiae , which can also be maintained as a BAC in E. coli , two self-replicating S. cerevisiae / E. coli shuttle vectors, pBelo-CEN-URA and pBelo-CEN-HYG, were constructed. .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG.

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene. .. Additional plasmids with early N4 promoters P1 (pKMK63), P2 (pKMK64), or P3 (pKMK65); four mutant P2 promoters (–10G to T, pKMK66; –11G to T, pKMK67; –12A to T, pKMK68; and +1C to T, pKMK69); and a control lacking the promoter (pKMK62) were constructed.

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein
    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer. .. H-NS was added in increasing concentrations to DNA constructs and incubated for 6 min at 37 °C before the addition of restriction enzymes.

    Incubation:

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein
    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer. .. H-NS was added in increasing concentrations to DNA constructs and incubated for 6 min at 37 °C before the addition of restriction enzymes.

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *
    Article Snippet: .. SDS was then sequestered from the samples by the addition of 1.8% Triton X-100 (Fisher), and the samples were incubated at 37 °C for 1 h. The samples were digested with BglII and/or ApaLI restriction enzyme (New England Biolabs) at 37 °C for 16 h. Restriction enzymes were inactivated by the addition of 1.6% SDS and further incubation at 65 °C for 20 min. .. Samples were diluted with T4 DNA ligase buffer (New England Biolabs) to achieve ∼3 ng of DNA/μl, and then 200 units of T4 DNA ligase (New England Biolabs) were added and incubated for 4 h at 16 °C.

    Activity Assay:

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: .. Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene. .. The resulting plasmid, pIK1, was used to determine in vivo activity of N4 mini-vRNAP variants.

    Expressing:

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: The resulting plasmid contains CEN4 cloned in between S. cerevisiae URA3 and a hygromycin-resistance expression cassette ( HygR ). .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG.

    Transformation Assay:

    Article Title: Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates
    Article Snippet: These oligonucleotides were annealed to a short complementary primer 5′-CAACAAGTTTCTGCGGCCGC -3′, treated with the large Klenow fragment to generate double-stranded DNA, double-digested with restriction endonucleases ApaLI and NotI (Italic) (NEB, Ipswich, SFK, UK). .. The final constructions were used for transformation of electrocompetent E. coli TG1 cells and tetracycline-resistant colonies were then selected.

    High Performance Liquid Chromatography:

    Article Title: The HsRAD51B-HsRAD51C stabilizes the HsRAD51 nucleoprotein filament
    Article Snippet: For surface plasmon resonance (SPR) analysis, a 5’-biotinylated oligo dT50 was used as ssDNA and for dsDNA 5’-biotinylated 50-mer 5’-TCG AGA GGG TAA ACC ACA-ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed with its complement and purified by HPLC on a Gen-Pak FAX column (Waters). .. RFIII DNA was obtained by linearizing RFI ϕX174 with ApaLI restriction enzyme (NEB).

    Southern Blot:

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: The purified DNA was subjected to Southern blot analysis. .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: Paragraph title: Detection of gene targeting events by Southern blot analysis ... After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Ligation:

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: MNase nucleosome mapping and restriction enzyme accessibility assays Micrococcal nuclease (MNase) digestion and ligation-mediated PCR (LM-PCR) were performed as described ( ). .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells
    Article Snippet: Paragraph title: Reverse transcription-polymerase chain reaction (RT-PCR) ... ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ).

    Sequencing:

    Article Title: Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule *
    Article Snippet: The resultant product, as well as pENTR containing segment one, was then digested with ApaLI (New England Biolabs, Ipswich, MA) and ligated together to yield the final full-length wnk cDNA in pENTR. .. The cDNA was sequenced in its entirety, and the sequence has been deposited in GenBankTM .

    Recombinant:

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: E. coli strain BL21 ( F – ompT hsd SB r B– m B– ) was used for the purification of recombinant proteins. .. Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene.

    In Vivo:

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: .. Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene. .. The resulting plasmid, pIK1, was used to determine in vivo activity of N4 mini-vRNAP variants.

    Mutagenesis:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: .. The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA). .. After digestion, the PCR products were electrophoresed on a 2% agarose gel and visualized by UltraPowerTM DNA Dye solution (Gelex International, Tokyo, Japan).

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: Bacterial Strains and Plasmids — E. coli strain DH5α ( supE44 Δ lacU169 (φ80 lacZ Δ M15 ) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ) was used to determine the in vivo activity of wild-type and mutant mini-vRNAP enzymes. .. Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene.

    Isolation:

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min. .. The purified genomic DNA was re-digested with 100 U of ApaI completely (for first digestion with XhoI, PstI and SmaI) or 100 U of PstI (for first digestion with SpeI and ApaLI).

    Size-exclusion Chromatography:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: Thermal cycling conditions were initial denaturation at 95°C for 1 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 61°C for 20 sec, and extension at 72°C for 20 sec. .. The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA).

    Purification:

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: The purified DNA was subjected to Southern blot analysis. .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: E. coli strain BL21 ( F – ompT hsd SB r B– m B– ) was used for the purification of recombinant proteins. .. Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene.

    Article Title: The HsRAD51B-HsRAD51C stabilizes the HsRAD51 nucleoprotein filament
    Article Snippet: For surface plasmon resonance (SPR) analysis, a 5’-biotinylated oligo dT50 was used as ssDNA and for dsDNA 5’-biotinylated 50-mer 5’-TCG AGA GGG TAA ACC ACA-ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed with its complement and purified by HPLC on a Gen-Pak FAX column (Waters). .. RFIII DNA was obtained by linearizing RFI ϕX174 with ApaLI restriction enzyme (NEB).

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: Detection of gene targeting events by Southern blot analysis Chromosomal DNA was purified according to a published method ( ). .. After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Article Title: Role of the conserved lysine within the Walker A motif of human DMC1
    Article Snippet: The ϕX174 replicative form I was digest with ApaLI (New England Biolabs) to linearize the DNA. .. The supercoiled pBluescript DNA was purified using a commercially available kit (Qiagen).

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *
    Article Snippet: SDS was then sequestered from the samples by the addition of 1.8% Triton X-100 (Fisher), and the samples were incubated at 37 °C for 1 h. The samples were digested with BglII and/or ApaLI restriction enzyme (New England Biolabs) at 37 °C for 16 h. Restriction enzymes were inactivated by the addition of 1.6% SDS and further incubation at 65 °C for 20 min. .. This was followed by the addition of 10 μg of RNase/ml, and the DNA was purified by phenol/chloroform extraction.

    Polymerase Chain Reaction:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: .. The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA). .. After digestion, the PCR products were electrophoresed on a 2% agarose gel and visualized by UltraPowerTM DNA Dye solution (Gelex International, Tokyo, Japan).

    Article Title: Hypotonicity Stimulates Potassium Flux through the WNK-SPAK/OSR1 Kinase Cascade and the Ncc69 Sodium-Potassium-2-Chloride Cotransporter in the Drosophila Renal Tubule *
    Article Snippet: PCR was performed with primers W5024D and W6483U, revealing an alternatively spliced exon within the 5′-UTR as well as redefining the splicing events in the 5′-UTR and suggesting a “new” start codon. .. The resultant product, as well as pENTR containing segment one, was then digested with ApaLI (New England Biolabs, Ipswich, MA) and ligated together to yield the final full-length wnk cDNA in pENTR.

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: MNase nucleosome mapping and restriction enzyme accessibility assays Micrococcal nuclease (MNase) digestion and ligation-mediated PCR (LM-PCR) were performed as described ( ). .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells
    Article Snippet: .. ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ). .. Because the ApaLI site in XBP-1 mRNA is spliced out upon activation, activated XBP-1 shows one large band after ApaLI digestion, while inactivated XBP-1 shows two ApaLI-digested bands.

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer. .. The 393-bp AAVS1 -specific probe was obtained by PCR amplification of human chromosomal DNA using 0.012 U/µl Phusion High-Fidelity DNA polymerase (Finnzymes), 200 µM deoxynucleoside triphosphates (dNTPs; Fermentas), 1× GC buffer (Finnzymes) plus 0.2 µM of primers #633 (5′-GGTCCCCAGCATGTCTTCCTA-3′) and #634 (5′-CTCCCGAACCTCAGATCTCC-3′).

    Gel Extraction:

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer. .. The resulting DNA fragment was purified after agarose gel electrophoresis with the aid of the QIAEX II gel extraction kit (Qiagen).

    Concentration Assay:

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein
    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer. .. The reactions were stopped by the addition of 50 mm EDTA and 0.2% SDS (final concentration).

    SPR Assay:

    Article Title: The HsRAD51B-HsRAD51C stabilizes the HsRAD51 nucleoprotein filament
    Article Snippet: For surface plasmon resonance (SPR) analysis, a 5’-biotinylated oligo dT50 was used as ssDNA and for dsDNA 5’-biotinylated 50-mer 5’-TCG AGA GGG TAA ACC ACA-ATT ATT GAT ATA AAA TAG TTT TGG GTA GGC GA was annealed with its complement and purified by HPLC on a Gen-Pak FAX column (Waters). .. RFIII DNA was obtained by linearizing RFI ϕX174 with ApaLI restriction enzyme (NEB).

    Plasmid Preparation:

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG. .. To construct BAC6-VH 3-11, initially two fragments, a 115-kb NotI-PmeI and a 110-kb RsrII-SgrAI, were cut out from the BAC clone 3054M17 CITB.

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene. .. The resulting plasmid, pIK1, was used to determine in vivo activity of N4 mini-vRNAP variants.

    Article Title: Profiling the Extended Cleavage Specificity of the House Dust Mite Protease Allergens Der p 1, Der p 3 and Der p 6 for the Prediction of New Cell Surface Protein Substrates
    Article Snippet: 4.2 Construction of Phage Substrate Libraries Three phage substrate libraries were created using the fd-Tet-DOG 1 vector based on previously described protocols [ , , ]. .. These oligonucleotides were annealed to a short complementary primer 5′-CAACAAGTTTCTGCGGCCGC -3′, treated with the large Klenow fragment to generate double-stranded DNA, double-digested with restriction endonucleases ApaLI and NotI (Italic) (NEB, Ipswich, SFK, UK).

    Software:

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein
    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer. .. The samples containing reaction products were loaded on agarose gels, and the intensities of individual DNA bands were analyzed using Gel analysis option on ImageJ software.

    Agarose Gel Electrophoresis:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA). .. After digestion, the PCR products were electrophoresed on a 2% agarose gel and visualized by UltraPowerTM DNA Dye solution (Gelex International, Tokyo, Japan).

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: .. After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer. .. Next, the DNA was transferred by capillary action to an Amersham Hybond-XL membrane (GE Healthcare) using a standard Southern blot technique.

    Activation Assay:

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells
    Article Snippet: ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ). .. Because the ApaLI site in XBP-1 mRNA is spliced out upon activation, activated XBP-1 shows one large band after ApaLI digestion, while inactivated XBP-1 shows two ApaLI-digested bands.

    BAC Assay:

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: To convert a linear YAC into a cYAC or to assemble DNA fragments with overlapping ends into a single cYAC in S. cerevisiae , which can also be maintained as a BAC in E. coli , two self-replicating S. cerevisiae / E. coli shuttle vectors, pBelo-CEN-URA and pBelo-CEN-HYG, were constructed. .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG.

    Lysis:

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *
    Article Snippet: Cross-linked cells were washed with ice-cold phosphate-buffered saline and lysed for 1 h with ice-cold lysis buffer containing 10 m m Tris (pH 8.0), 10 m m NaCl, 0.2% Nonidet P-40, and 1 m m dithiothreitol. .. SDS was then sequestered from the samples by the addition of 1.8% Triton X-100 (Fisher), and the samples were incubated at 37 °C for 1 h. The samples were digested with BglII and/or ApaLI restriction enzyme (New England Biolabs) at 37 °C for 16 h. Restriction enzymes were inactivated by the addition of 1.6% SDS and further incubation at 65 °C for 20 min.

    Variant Assay:

    Article Title: Identification of Bacteriophage N4 Virion RNA Polymerase-Nucleic Acid Interactions in Transcription Complexes *
    Article Snippet: Reporter Plasmids to Determine in Vivo Activity of Mini-vRNAP —A fragment flanked by ApaLI- and BamHI restriction sites that includes vRNAP promoter P2 (–19 to +2) followed by the lacZ ′ gene from M13mp18 was inserted into ApaLI and BamHI-digested pACYC177 (New England Biolabs), disrupting the β-lactamase gene. .. In Vivo Activity of Mini-vRNAP Variants and Mutant Promoters — E. coli strain DH5α cells carrying pIK1 and a plasmid encoding a mini-vRNAP variant (see below) were grown in Lennox Broth base medium containing 100 μg/ml ampicillin and 50 μl/ml kanamycin (Sigma) until they reached a density of ∼109 cells/ml.

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    New England Biolabs apali
    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with <t>BamHI-HindIII</t> ( A ), BamHI-EcoRI ( B ), and <t>ApaLI</t> ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
    Apali, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Journal: Nucleic Acids Research

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    doi: 10.1093/nar/gkp643

    Figure Lengend Snippet: Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Techniques: Southern Blot, Clone Assay, Transfection, Derivative Assay, Plasmid Preparation, Molecular Weight, Amplification, Binding Assay, Marker

    PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Journal: Nucleic Acids Research

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    doi: 10.1093/nar/gkp643

    Figure Lengend Snippet: PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Techniques: Polymerase Chain Reaction, Genetically Modified, Stable Transfection, Nested PCR, Clone Assay, Derivative Assay, Transfection, Plasmid Preparation, Labeling, Amplification, Negative Control, Marker, Molecular Weight, Agarose Gel Electrophoresis

    Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Journal: International Journal of Oncology

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

    doi: 10.3892/ijo.2014.2804

    Figure Lengend Snippet: Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Article Snippet: ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ).

    Techniques: Activation Assay, Western Blot, Incubation, Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII

    Journal: The Journal of Biological Chemistry

    Article Title: Sp1 Regulates Chromatin Looping between an Intronic Enhancer and Distal Promoter of the Human Heme Oxygenase-1 Gene in Renal Cells *

    doi: 10.1074/jbc.M109.058586

    Figure Lengend Snippet: Confirmation of chromatin looping by 3C assay with BglII and/or ApaLI restriction enzyme. A , schematic showing potential interaction between the fragments containing HS-2 region and the internal enhancer ( square ) and transcription factor(s) ( TF ). B , BglII

    Article Snippet: SDS was then sequestered from the samples by the addition of 1.8% Triton X-100 (Fisher), and the samples were incubated at 37 °C for 1 h. The samples were digested with BglII and/or ApaLI restriction enzyme (New England Biolabs) at 37 °C for 16 h. Restriction enzymes were inactivated by the addition of 1.6% SDS and further incubation at 65 °C for 20 min.

    Techniques: