pbr322 plasmid Search Results


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  • 98
    New England Biolabs pbr322 vector
    Pbr322 Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbr322 vector/product/New England Biolabs
    Average 98 stars, based on 71 article reviews
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    95
    ATCC pbr322 plasmid
    Pbr322 Plasmid, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 19 article reviews
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    95
    Thermo Fisher pbr322 plasmid dna
    GSH potentiates kinamycin F-induced <t>DNA</t> nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of <t>pBR322</t> plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.
    Pbr322 Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 7683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher plasmids pbr322
    Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and <t>pBR322</t> and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.
    Plasmids Pbr322, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATUM pbr322 plasmid dna
    Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). <t>DNA</t> refers to untreated plasmid <t>pBR322.</t> a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.
    Pbr322 Plasmid Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa pbr322 plasmid dna
    The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated <t>DNA</t> relaxation indifferent concentration. Lane 1, supercoiled <t>pBR322</t> plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.
    Pbr322 Plasmid Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    ICN Biomedicals pbr322 plasmid dna
    Electrophoretograms applying to the incubated mixtures of <t>pBR322</t> plasmid <t>DNA</t> and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.
    Pbr322 Plasmid Dna, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore plasmid pbr322
    HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid <t>pBR322</t> (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.
    Plasmid Pbr322, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore pbr322 plasmid dna
    Electrophoresis of <t>DNA</t> plasmid <t>pBR322</t> incubated in 5 mM Tris/HCl buffer for 20 h at 37°C with cisplatin, CQDP, complex 1 and complex 6 . Lanes 1 and 13: molecular marker; lane 2: DNA pBR322; lane 3: DNA treated with 0.5 eq. of cisplatin; lanes
    Pbr322 Plasmid Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATCC pbr322 dunlop plasmid
    The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the <t>pBR322-Dunlop</t> or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.
    Pbr322 Dunlop Plasmid, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Stratagene plasmid pbr322
    The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the <t>pBR322-Dunlop</t> or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.
    Plasmid Pbr322, supplied by Stratagene, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Mannheim supercoiled pbr322 plasmid dna
    The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the <t>pBR322-Dunlop</t> or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.
    Supercoiled Pbr322 Plasmid Dna, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Journal: Free radical biology & medicine

    Article Title: Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F

    doi: 10.1016/j.freeradbiomed.2007.07.005

    Figure Lengend Snippet: GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Article Snippet: Thioglo-1 was from EMD Biosciences (Mississauga, Canada), kDNA was obtained from TopoGEN (Columbus, OH) and pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington, ON, Canada).

    Techniques: Staining, Plasmid Preparation

    The DNA cross-linking effects of the hybrids, cisplatin and chlorambucil on linearized pBR322 DNA. This fluorescent image of the ethidium bromide-stained gel shows that the bifunctional alkylating agent chlorambucil control and cisplatin cross-linked DNA while the hybrids only slightly increased cross linking. All agents were tested at a concentration of 10 µM. Control 1 was not heated and thus consisted of only double stranded DNA. Control 2 and the other drug-treated samples were heated to 90 °C for 5 min to cause strand separation and then rapidly cooled. The strands that were cross-linked by drug treatment recombined to form double-strand DNA, whereas those that were not cross-linked, did not recombine. The amount of DNA in each well was the same. The band intensity of the single stranded DNA is less than that of double strand DNA because of reduced binding of ethidium bromide.

    Journal: Bioorganic & medicinal chemistry

    Article Title: Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

    doi: 10.1016/j.bmc.2014.09.014

    Figure Lengend Snippet: The DNA cross-linking effects of the hybrids, cisplatin and chlorambucil on linearized pBR322 DNA. This fluorescent image of the ethidium bromide-stained gel shows that the bifunctional alkylating agent chlorambucil control and cisplatin cross-linked DNA while the hybrids only slightly increased cross linking. All agents were tested at a concentration of 10 µM. Control 1 was not heated and thus consisted of only double stranded DNA. Control 2 and the other drug-treated samples were heated to 90 °C for 5 min to cause strand separation and then rapidly cooled. The strands that were cross-linked by drug treatment recombined to form double-strand DNA, whereas those that were not cross-linked, did not recombine. The amount of DNA in each well was the same. The band intensity of the single stranded DNA is less than that of double strand DNA because of reduced binding of ethidium bromide.

    Article Snippet: Briefly, the pBR322 DNA (1.2 µg,) was linearized at a single restriction site with HindIII restriction enzyme (20 units, Invitrogen) in 20 µL of buffer (50 mM Tris/1 mM MgCl2 /5 mM NaCl, pH 8.0) and purified by a Wizard SV Gel and PCR Clean-up System (Promega).

    Techniques: Staining, Concentration Assay, Binding Assay

    Concentration dependent effect of the hybrids, the piperazine advanced intermediates 15 and 16 , and etoposide on the topoisomerase IIα-mediated relaxation and cleavage of supercoiled pBR322 plasmid DNA to produce linear DNA. These fluorescent images of ethidium bromide-stained gels show that topoisomerase IIα (+Topo IIα) converted supercoiled (SC) pBR322 DNA to relaxed (RLX) DNA. In this assay the relaxed DNA runs slightly ahead of the supercoiled DNA because the gel was run in the presence of ethidium bromide. Topoisomerase IIα was present in the reaction mixture in all but the lanes marked pBR322. The etoposide positive controls produced significant amounts of linear DNA (LIN). A small amount of nicked circular (NC), which may arise from strand breakage during isolation, is normally present in pBR322 DNA. The results were typical of experiments carried out on 2 different days.

    Journal: Bioorganic & medicinal chemistry

    Article Title: Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin-N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

    doi: 10.1016/j.bmc.2014.09.014

    Figure Lengend Snippet: Concentration dependent effect of the hybrids, the piperazine advanced intermediates 15 and 16 , and etoposide on the topoisomerase IIα-mediated relaxation and cleavage of supercoiled pBR322 plasmid DNA to produce linear DNA. These fluorescent images of ethidium bromide-stained gels show that topoisomerase IIα (+Topo IIα) converted supercoiled (SC) pBR322 DNA to relaxed (RLX) DNA. In this assay the relaxed DNA runs slightly ahead of the supercoiled DNA because the gel was run in the presence of ethidium bromide. Topoisomerase IIα was present in the reaction mixture in all but the lanes marked pBR322. The etoposide positive controls produced significant amounts of linear DNA (LIN). A small amount of nicked circular (NC), which may arise from strand breakage during isolation, is normally present in pBR322 DNA. The results were typical of experiments carried out on 2 different days.

    Article Snippet: Briefly, the pBR322 DNA (1.2 µg,) was linearized at a single restriction site with HindIII restriction enzyme (20 units, Invitrogen) in 20 µL of buffer (50 mM Tris/1 mM MgCl2 /5 mM NaCl, pH 8.0) and purified by a Wizard SV Gel and PCR Clean-up System (Promega).

    Techniques: Concentration Assay, Plasmid Preparation, Staining, Produced, Isolation

    Effect of neocuproine (500 μM) on KQ -Cu(II) (300 μM) induced pBR322 DNA breakage. 500 ng pBR322 DNA was used in each lane. (a) pBR322 DNA; (b) DNA + Cu(II); (c) DNA + Cu(II) + neocuproine; (d) DNA + Cu(II) + KQ (100 μM) + neocuproine. OC, open circular DNA; SC, supercoiled DNA. The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Journal: Molecules

    Article Title: Kazinol Q from Broussonetia kazinoki Enhances Cell Death Induced by Cu(ll) through Increased Reactive Oxygen Species

    doi: 10.3390/molecules16043212

    Figure Lengend Snippet: Effect of neocuproine (500 μM) on KQ -Cu(II) (300 μM) induced pBR322 DNA breakage. 500 ng pBR322 DNA was used in each lane. (a) pBR322 DNA; (b) DNA + Cu(II); (c) DNA + Cu(II) + neocuproine; (d) DNA + Cu(II) + KQ (100 μM) + neocuproine. OC, open circular DNA; SC, supercoiled DNA. The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Article Snippet: Supercoiled pBR322 plasmid DNA was purchased from ABgene House (Epsom, Surrey, UK).

    Techniques:

    Effect of active oxygen scavengers on KQ -Cu(II)-mediated DNA breakage: (a) DNA alone; (b) DNA + Cu(II) (300 μM) + KQ (300 μM); (c-e) same as (b) with KI (750 μM), SOD (0.1 mg/mL), Catalase (0.1 mg/mL), respectively. OC, open circular DNA; SC, supercoiled DNA. The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Journal: Molecules

    Article Title: Kazinol Q from Broussonetia kazinoki Enhances Cell Death Induced by Cu(ll) through Increased Reactive Oxygen Species

    doi: 10.3390/molecules16043212

    Figure Lengend Snippet: Effect of active oxygen scavengers on KQ -Cu(II)-mediated DNA breakage: (a) DNA alone; (b) DNA + Cu(II) (300 μM) + KQ (300 μM); (c-e) same as (b) with KI (750 μM), SOD (0.1 mg/mL), Catalase (0.1 mg/mL), respectively. OC, open circular DNA; SC, supercoiled DNA. The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Article Snippet: Supercoiled pBR322 plasmid DNA was purchased from ABgene House (Epsom, Surrey, UK).

    Techniques:

    DNA strand scission by KQ . pBR322 plasmid DNA (500 ng) was incubated for 30 min at 37 °C in the presence of following additives: (a) 300 μM Cu(II); (b) 300 μM KQ ; (c) 300 μM KQ + 300 μM Cu(II); (d) 200 μM KQ + 200 μM Cu(II); (e) 100 μM KQ + 100 μM Cu(II); (f) 50 μM KQ + 50 μM Cu(II); (g) 25 μM KQ + 25 μM Cu(II).OC, open circular DNA; SC, supercoiled DNA.The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Journal: Molecules

    Article Title: Kazinol Q from Broussonetia kazinoki Enhances Cell Death Induced by Cu(ll) through Increased Reactive Oxygen Species

    doi: 10.3390/molecules16043212

    Figure Lengend Snippet: DNA strand scission by KQ . pBR322 plasmid DNA (500 ng) was incubated for 30 min at 37 °C in the presence of following additives: (a) 300 μM Cu(II); (b) 300 μM KQ ; (c) 300 μM KQ + 300 μM Cu(II); (d) 200 μM KQ + 200 μM Cu(II); (e) 100 μM KQ + 100 μM Cu(II); (f) 50 μM KQ + 50 μM Cu(II); (g) 25 μM KQ + 25 μM Cu(II).OC, open circular DNA; SC, supercoiled DNA.The percentage of OC DNA as compared to total DNA is individually shown below each lane.

    Article Snippet: Supercoiled pBR322 plasmid DNA was purchased from ABgene House (Epsom, Surrey, UK).

    Techniques: Plasmid Preparation, Incubation

    Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and pBR322 and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

    doi: 10.1016/j.sjbs.2019.11.017

    Figure Lengend Snippet: Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and pBR322 and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.

    Article Snippet: The following plasmids pBR322 and pUC18, and λDNA were obtained from ThermoFisher Scientific, treated in the same way and used as controls.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Countercurrent Chromatography

    Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Digital PCR, Plasmid Preparation

    PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Expressing

    Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Plasmid Preparation, Concentration Assay, Digital PCR, Labeling

    Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Digital PCR, Serial Dilution, Labeling

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Journal: Methods in cell biology

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    doi: 10.1016/bs.mcb.2015.01.021

    Figure Lengend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Article Snippet: pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol.

    Techniques: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

    Cell-free setup for visualizing SopAB-mediated dynamics of plasmids or beads on a DNA carpet. (A) SopA and SopB proteins are preincubated with plasmid or bead cargo bearing the sopC -centromere site in Sop buffer containing ATP. The sample is then flowed into the DNA-carpeted flow cell. Flow is stopped to visualize how SopA and SopB mediate cargo dynamics on the DNA carpet. (B) Excitation lasers are directed onto the flow cell at an angle that allows total internal reflection (TIRF). The evanescent wave (blue haze) produced by the incident and reflected light propagates into the flow cell ~100 nm. SopA–GFP (green) is covisualized with either SopBeAlexa647, or one of two possible cargos labeled with Alexa647, the pBR322:: sopC plasmid or sopC -coated beads. (i) ATP-bound SopA binds the DNA carpet and tethers the plasmid by SopA–SopB interaction, (ii) until SopB locally stimulates the removal of SopA. (iii) Once all SopA anchor points are removed, the plasmid is released and is no longer observable via TIRFM. A magnet above the flow cell surface confines sopC -coated magnetic beads on the DNA carpet, which supports persistent SopA removal and directed bead movement. (See color plate)

    Journal: Methods in cell biology

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    doi: 10.1016/bs.mcb.2015.01.021

    Figure Lengend Snippet: Cell-free setup for visualizing SopAB-mediated dynamics of plasmids or beads on a DNA carpet. (A) SopA and SopB proteins are preincubated with plasmid or bead cargo bearing the sopC -centromere site in Sop buffer containing ATP. The sample is then flowed into the DNA-carpeted flow cell. Flow is stopped to visualize how SopA and SopB mediate cargo dynamics on the DNA carpet. (B) Excitation lasers are directed onto the flow cell at an angle that allows total internal reflection (TIRF). The evanescent wave (blue haze) produced by the incident and reflected light propagates into the flow cell ~100 nm. SopA–GFP (green) is covisualized with either SopBeAlexa647, or one of two possible cargos labeled with Alexa647, the pBR322:: sopC plasmid or sopC -coated beads. (i) ATP-bound SopA binds the DNA carpet and tethers the plasmid by SopA–SopB interaction, (ii) until SopB locally stimulates the removal of SopA. (iii) Once all SopA anchor points are removed, the plasmid is released and is no longer observable via TIRFM. A magnet above the flow cell surface confines sopC -coated magnetic beads on the DNA carpet, which supports persistent SopA removal and directed bead movement. (See color plate)

    Article Snippet: pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol.

    Techniques: Plasmid Preparation, Flow Cytometry, Produced, Labeling, Magnetic Beads

    Complementation of the lpp mutant of Y. pestis CO92 with the htrA gene restores intracellular survival in macrophages. Intracellular survival of WT with pBR322, Δ lpp mutant with pBR322, and the Δ lpp with pBR322 htrA was determined by infecting RAW 264.7 murine macrophages with an MOI of 1 for 45 minutes, followed by a 60-minute gentamicin wash, and plating the surviving intracellular bacteria at 4, 8, and 12 hours. The Δ lpp mutant with pBR322 htrA has a significant increase in percent survival compared to the Δ lpp mutant with pBR322 alone, as determined by ANOVA and Holm-Sidak method.

    Journal: Comparative and Functional Genomics

    Article Title: Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures

    doi: 10.1155/2010/342168

    Figure Lengend Snippet: Complementation of the lpp mutant of Y. pestis CO92 with the htrA gene restores intracellular survival in macrophages. Intracellular survival of WT with pBR322, Δ lpp mutant with pBR322, and the Δ lpp with pBR322 htrA was determined by infecting RAW 264.7 murine macrophages with an MOI of 1 for 45 minutes, followed by a 60-minute gentamicin wash, and plating the surviving intracellular bacteria at 4, 8, and 12 hours. The Δ lpp mutant with pBR322 htrA has a significant increase in percent survival compared to the Δ lpp mutant with pBR322 alone, as determined by ANOVA and Holm-Sidak method.

    Article Snippet: The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and digested using the restriction endonucleases Bam HI and Sal I (New England BioLabs, Ipswich, MA) to prepare the fragment for ligation into pBR322 vector (Fermentas, Glen Burnie, MD) at the appropriate restriction enzyme sites.

    Techniques: Mutagenesis

    Topoisomerase activity of wild-type and truncated Int SSV2 proteins. Wild-type or truncated Int SSV2 was incubated for 4 h at 65°C with negatively supercoiled pBR322 DNA (0.6 μg). The samples were treated with proteinase K and SDS and subjected

    Journal: Journal of Virology

    Article Title: Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions

    doi: 10.1128/JVI.01637-15

    Figure Lengend Snippet: Topoisomerase activity of wild-type and truncated Int SSV2 proteins. Wild-type or truncated Int SSV2 was incubated for 4 h at 65°C with negatively supercoiled pBR322 DNA (0.6 μg). The samples were treated with proteinase K and SDS and subjected

    Article Snippet: First, we tested the cleavage/religation or topoisomerase activity of the full-length and deletion mutant proteins by using negatively supercoiled plasmid pBR322.

    Techniques: Activity Assay, Incubation

    ( A ) Scanning image of intercalated pBR322 DNA fragments adsorbed on PLL-coated glass slides (25 × 25 µm 2 , pH 6, vertical scanning direction). Brightness of the molecules measured in PE. Adsorbed molecules cover a larger area than diffusing ones (small lines in the upper section of the image). ( B ) Histogram of the brightness of the adsorbed pBR322 DNA fragments, background of 12 PE substracted. Bin width 2 PE. Histogram was fitted with Gaussian curve (black line). The reduced χ 2 of the fit is 0.75.

    Journal: Nucleic Acids Research

    Article Title: Sizing of single fluorescently stained DNA fragments by scanning microscopy

    doi: 10.1093/nar/gng138

    Figure Lengend Snippet: ( A ) Scanning image of intercalated pBR322 DNA fragments adsorbed on PLL-coated glass slides (25 × 25 µm 2 , pH 6, vertical scanning direction). Brightness of the molecules measured in PE. Adsorbed molecules cover a larger area than diffusing ones (small lines in the upper section of the image). ( B ) Histogram of the brightness of the adsorbed pBR322 DNA fragments, background of 12 PE substracted. Bin width 2 PE. Histogram was fitted with Gaussian curve (black line). The reduced χ 2 of the fit is 0.75.

    Article Snippet: 14 200 bp plasmid DNA was isolated from EcoRI cells with the Plasmid Mini Kit (Qiagen, Germany). m13mp18 RF1 DNA (7250 bp), the plasmids pBR322 DNA (4361 bp) and pUC19 DNA (2686 bp) were purchased from MBI Fermentas, Germany. m13mp18 RF1 DNA was digested at 36°C for 4 h by the restriction enzymes EcoRI and PagI (MBI Fermentas, Germany) into 2318 and 4932 bp fragments.

    Techniques:

    Gel electrophoresis of used DNA fragments (1) and an EcoRI and PagI digest of m13mp18 RF1 DNA (2), as described in Materials and Methods. ( 1 ) Lines: (a) 1985 bp linear DNA, (b) pUC19 plasmid DNA, (c) 4120 bp linear DNA, (d) pBR322 plasmid DNA, (e) m13mp18 RF1 DNA, (f) 14 000 bp plasmid DNA and (g) DNA molecular weight marker II [23 130, 9416, 6557, 4361, 2322, 2027 bp (Roche, Switzerland)]. ( 2 ) Lines: (a) m13mp18 RF1 DNA, (b) EcoRI/PagI digest of m13mp18 RF1 DNA, (c) DNA molecular weight marker II (Roche, Switzerland).

    Journal: Nucleic Acids Research

    Article Title: Sizing of single fluorescently stained DNA fragments by scanning microscopy

    doi: 10.1093/nar/gng138

    Figure Lengend Snippet: Gel electrophoresis of used DNA fragments (1) and an EcoRI and PagI digest of m13mp18 RF1 DNA (2), as described in Materials and Methods. ( 1 ) Lines: (a) 1985 bp linear DNA, (b) pUC19 plasmid DNA, (c) 4120 bp linear DNA, (d) pBR322 plasmid DNA, (e) m13mp18 RF1 DNA, (f) 14 000 bp plasmid DNA and (g) DNA molecular weight marker II [23 130, 9416, 6557, 4361, 2322, 2027 bp (Roche, Switzerland)]. ( 2 ) Lines: (a) m13mp18 RF1 DNA, (b) EcoRI/PagI digest of m13mp18 RF1 DNA, (c) DNA molecular weight marker II (Roche, Switzerland).

    Article Snippet: 14 200 bp plasmid DNA was isolated from EcoRI cells with the Plasmid Mini Kit (Qiagen, Germany). m13mp18 RF1 DNA (7250 bp), the plasmids pBR322 DNA (4361 bp) and pUC19 DNA (2686 bp) were purchased from MBI Fermentas, Germany. m13mp18 RF1 DNA was digested at 36°C for 4 h by the restriction enzymes EcoRI and PagI (MBI Fermentas, Germany) into 2318 and 4932 bp fragments.

    Techniques: Nucleic Acid Electrophoresis, Plasmid Preparation, Molecular Weight, Marker

    Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). DNA refers to untreated plasmid pBR322. a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Journal: Organometallics

    Article Title: Titanocene–Gold Complexes Containing N-Heterocyclic Carbene Ligands Inhibit Growth of Prostate, Renal, and Colon Cancers in Vitro

    doi: 10.1021/acs.organomet.6b00051

    Figure Lengend Snippet: Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). DNA refers to untreated plasmid pBR322. a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Article Snippet: Interactions of the New Compounds with Plasmid pBR322 (Gel Electrophoresis Mobility Shift Assay) Ten microliter aliquots of pBR322 plasmid DNA (20 μg/mL) in buffer (5 mM Tris/HCl, 50 mM NaClO4 , pH 7.39) were incubated with different concentrations of the compounds ( 4a – d , 5a – d , and cisplatin as control) (in the range 0.25 and 4.0 metal complex to DNA bp) at 37 °C for 20 h in the dark.

    Techniques: Electrophoresis, Mobility Shift, Plasmid Preparation

    Electrophoresis mobility shift assays for cisplatin and compounds 1 – 5 (see Experimental Section for details). DNA refers to untreated plasmid pBR322. A, B, C, and D correspond to metal/DNAbp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Journal: Journal of Medicinal Chemistry

    Article Title: Cyclometalated Iminophosphorane Gold(III) and Platinum(II) Complexes. A Highly Permeable Cationic Platinum(II) Compound with Promising Anticancer Properties

    doi: 10.1021/acs.jmedchem.5b00427

    Figure Lengend Snippet: Electrophoresis mobility shift assays for cisplatin and compounds 1 – 5 (see Experimental Section for details). DNA refers to untreated plasmid pBR322. A, B, C, and D correspond to metal/DNAbp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Article Snippet: Interaction of Compounds 1 – 5 and Cisplatin with Plasmid (pBR322) DNA by Electrophoresis (Mobility Shift Assay) First, 10 μL aliquots of pBR322 plasmid DNA (20 μg/mL) in buffer (5 mM Tris-HCl, 50 mM NaClO4 , pH 7.39) were incubated with different concentrations of the compounds 1 – 5 (in the range 0.25–2.0 metal complex:DNAbp) at 37 °C for 20 h in the dark.

    Techniques: Electrophoresis, Mobility Shift, Plasmid Preparation

    The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated DNA relaxation indifferent concentration. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.

    Journal: Scientific Reports

    Article Title: The gene expression profiles in response to 102 traditional Chinese medicine (TCM) components: a general template for research on TCMs

    doi: 10.1038/s41598-017-00535-8

    Figure Lengend Snippet: The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated DNA relaxation indifferent concentration. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.

    Article Snippet: Compound concentrations, pBR322 plasmid DNA (0.25 μg) and 1 unit of TopI (TaKaRa Biotechnology Co., Ltd., Dalian) were combined in a final volume of 20 μL buffer (35 mM pH 8.0 Tris-HCl, 72 mM KCl, 5 mM MgCl2 , 5 mM dithiothreitol, 5 mM spermidine, 0.1% bovine serum albumin).

    Techniques: Concentration Assay, Plasmid Preparation, Inhibition, Activity Assay

    Inhibition of DNA-triggered aggregation of oxidized SOD1 proteins in 20 mM Tris-HCl buffer (pH 7.4). A) Effect of ionic strength was observed by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA at 37°C for 2 h in the presence of 0–800 mM NaCl. Here, the inhibition degree of aggregation is expressed by [(RALS) 0 – (RALS) NaCl ]/(RALS) 0 ×100%, (RALS) 0 and (RALS) NaCl represent RALS values in the absence and in the presence of NaCl at each concentration, respectively. B) The inhibitory effect of GdmCl was monitored by RALS measurements. Reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA were incubated at 37°C for 2 h in the presence of 0–6 M GdmCl. The disaggregation degree of aggregation is expressed as (A). C) The disaggregation and inhibition of aggregates was monitored by agarose gel electrophoresis. The aggregates were produced by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 15 µM pBR322 DNA for 24 h at 37°C, and re-incubated for 1 min with 0–6 M GdmCl before loading onto gels. D) The inhibition of aggregation caused by EDTA was monitored by agarose gel electrophoresis. 4 µM SOD1 and 2 mM ascorbate were incubated for 2 h at 37°C with 15 µM pBR322 DNA in the presence of 0–100 mM EDTA before loading onto gels.

    Journal: PLoS ONE

    Article Title: DNA-Triggered Aggregation of Copper, Zinc Superoxide Dismutase in the Presence of Ascorbate

    doi: 10.1371/journal.pone.0012328

    Figure Lengend Snippet: Inhibition of DNA-triggered aggregation of oxidized SOD1 proteins in 20 mM Tris-HCl buffer (pH 7.4). A) Effect of ionic strength was observed by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA at 37°C for 2 h in the presence of 0–800 mM NaCl. Here, the inhibition degree of aggregation is expressed by [(RALS) 0 – (RALS) NaCl ]/(RALS) 0 ×100%, (RALS) 0 and (RALS) NaCl represent RALS values in the absence and in the presence of NaCl at each concentration, respectively. B) The inhibitory effect of GdmCl was monitored by RALS measurements. Reactions containing 4 µM SOD1, 2 mM Asc, and 7.5 µM ctDNA were incubated at 37°C for 2 h in the presence of 0–6 M GdmCl. The disaggregation degree of aggregation is expressed as (A). C) The disaggregation and inhibition of aggregates was monitored by agarose gel electrophoresis. The aggregates were produced by incubating reactions containing 4 µM SOD1, 2 mM Asc, and 15 µM pBR322 DNA for 24 h at 37°C, and re-incubated for 1 min with 0–6 M GdmCl before loading onto gels. D) The inhibition of aggregation caused by EDTA was monitored by agarose gel electrophoresis. 4 µM SOD1 and 2 mM ascorbate were incubated for 2 h at 37°C with 15 µM pBR322 DNA in the presence of 0–100 mM EDTA before loading onto gels.

    Article Snippet: The plasmid pBR322 DNA and bacteriophage λDNA were purchased from TaKaRa, the ssDNA (24-nt, 5′-GGTCGGAGTCAACGGATTTGGTCG-3′ ) was purchased from Invitrogen.

    Techniques: Inhibition, Concentration Assay, Incubation, Agarose Gel Electrophoresis, Produced

    DNA damage protective effect of SBP-III-3. Lane 1, the native pBR322DNA; lanes 2, the DNA treated with FeSO 4 , H 2 O 2 and SBP-III-3 (3.0 mg/mL); lane 3, the DNA treated with FeSO 4 , H 2 O 2 , and ascorbic acid (1.0 mg/mL); lane 4, the DNA treated with FeSO 4 , H 2 O 2 , and SBP-III-3 (1.0 mg/mL); lane 5, the pBR322DNA treated with FeSO 4 and H 2 O 2 .

    Journal: Marine Drugs

    Article Title: Anti-Fatigue Effect by Peptide Fraction from Protein Hydrolysate of Croceine Croaker (Pseudosciaena crocea) Swim Bladder through Inhibiting the Oxidative Reactions including DNA Damage

    doi: 10.3390/md14120221

    Figure Lengend Snippet: DNA damage protective effect of SBP-III-3. Lane 1, the native pBR322DNA; lanes 2, the DNA treated with FeSO 4 , H 2 O 2 and SBP-III-3 (3.0 mg/mL); lane 3, the DNA treated with FeSO 4 , H 2 O 2 , and ascorbic acid (1.0 mg/mL); lane 4, the DNA treated with FeSO 4 , H 2 O 2 , and SBP-III-3 (1.0 mg/mL); lane 5, the pBR322DNA treated with FeSO 4 and H 2 O 2 .

    Article Snippet: Plasmid DNA (pBR322DNA) was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).

    Techniques:

    Electrophoretograms applying to the incubated mixtures of pBR322 plasmid DNA and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.

    Journal: Journal of Biomedical Science

    Article Title: Synthesis and activity of three new trinuclear platinums with cis-geometry for terminal metal centres

    doi: 10.1186/1423-0127-21-41

    Figure Lengend Snippet: Electrophoretograms applying to the incubated mixtures of pBR322 plasmid DNA and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.

    Article Snippet: Exactly, 1.5 μL of supplied pBR322 plasmid DNA in solution was added to solutions of the compounds at different concentrations ranging from 0.55 μM to 70 μM for QH4, QH8 and cisplatin.

    Techniques: Incubation, Plasmid Preparation

    Electrophotograms applying to the interaction of pBR322 plasmid DNA with increasing concentrations of QH4, QH7, QH8 and cisplatin. Lane B applied to untreated pBR322 plasmid DNA to serve as a control, lanes 1 to 8 applied to plasmid DNA interacted with increasing concentrations of the compounds and cisplatin. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12 μM, lane 2 : 0.23 μM, lane 3: 0.47 μM, lane 4: 0.94 μM, lane 5:1.88 μM, lane 6: 3.75 μM, lane 7: 7.50 μM and lane 8: 15.00 μM.

    Journal: Journal of Biomedical Science

    Article Title: Synthesis and activity of three new trinuclear platinums with cis-geometry for terminal metal centres

    doi: 10.1186/1423-0127-21-41

    Figure Lengend Snippet: Electrophotograms applying to the interaction of pBR322 plasmid DNA with increasing concentrations of QH4, QH7, QH8 and cisplatin. Lane B applied to untreated pBR322 plasmid DNA to serve as a control, lanes 1 to 8 applied to plasmid DNA interacted with increasing concentrations of the compounds and cisplatin. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12 μM, lane 2 : 0.23 μM, lane 3: 0.47 μM, lane 4: 0.94 μM, lane 5:1.88 μM, lane 6: 3.75 μM, lane 7: 7.50 μM and lane 8: 15.00 μM.

    Article Snippet: Exactly, 1.5 μL of supplied pBR322 plasmid DNA in solution was added to solutions of the compounds at different concentrations ranging from 0.55 μM to 70 μM for QH4, QH8 and cisplatin.

    Techniques: Plasmid Preparation

    HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Inhibition, Activity Assay, Agarose Gel Electrophoresis, Electrophoresis, Staining, Plasmid Preparation, Thin Layer Chromatography

    HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Plasmid Preparation, Incubation, Staining

    Stimulation of topo IIα activity by HMGB1 is not due to the effect of HMGB1 on DNA topology. ( A ) HMGB1 protein and structure of HMGB1 domains A and B indicating positions of the mutated intercalating amino acid residues (in red frames). ( B ) Purity of HMGB1 and mutant as revealed by electrophoresis on an SDS–18% polyacrylamide gel and Coomassie blue R-250 staining. Lane 1, wild-type HMGB1; lane 2, HMGB1 mutant (F38A/F103A/I122A). A, alanine; F, phenylalanine; I, isoleucine. ( C ) Alanine mutagenesis of intercalating residues of HMGB1 abrogated the ability of HMGB1 to introduce supercoils into DNA by topoisomerase I. Closed-circular pBR322 plasmid DNA (∼9 nM) was incubated with wheat germ topoisomerase I in the absence or presence of HMGB1 or mutant (1, 2, 3 and 6 μM, left to right). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. (D ) Both wild-type HMGB1 and HMGB1 mutant can enhance decatenation of kDNA by human topo IIα. Decatenation of kDNA was carried out as in Figure 3 A. HMGB1 and mutant were at 1 μM, topo IIα was 7.5 and 15 nM. ( E ) HMGB1 does not stimulate decatenation of kDNA by E. coli topoisomerase IV. Decatenation of kDNA by E. coli topoisomerase IV (0.15 and 0.6 U) was carried out as detailed in the Materials and Methods section using HMGB1 or mutant (1 and 4 μM, left to right). The DNA samples in (C–E) were separated on 0.8% agarose gels without (C) or with (D–E) ethidium bromide in the course of electrophoresis as detailed in the Materials and Methods section. The gels are presented as negatives. WT, wild-type HMGB1; Mut, triple-mutant (F38A/F103A/I122A) of HMGB1.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: Stimulation of topo IIα activity by HMGB1 is not due to the effect of HMGB1 on DNA topology. ( A ) HMGB1 protein and structure of HMGB1 domains A and B indicating positions of the mutated intercalating amino acid residues (in red frames). ( B ) Purity of HMGB1 and mutant as revealed by electrophoresis on an SDS–18% polyacrylamide gel and Coomassie blue R-250 staining. Lane 1, wild-type HMGB1; lane 2, HMGB1 mutant (F38A/F103A/I122A). A, alanine; F, phenylalanine; I, isoleucine. ( C ) Alanine mutagenesis of intercalating residues of HMGB1 abrogated the ability of HMGB1 to introduce supercoils into DNA by topoisomerase I. Closed-circular pBR322 plasmid DNA (∼9 nM) was incubated with wheat germ topoisomerase I in the absence or presence of HMGB1 or mutant (1, 2, 3 and 6 μM, left to right). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. (D ) Both wild-type HMGB1 and HMGB1 mutant can enhance decatenation of kDNA by human topo IIα. Decatenation of kDNA was carried out as in Figure 3 A. HMGB1 and mutant were at 1 μM, topo IIα was 7.5 and 15 nM. ( E ) HMGB1 does not stimulate decatenation of kDNA by E. coli topoisomerase IV. Decatenation of kDNA by E. coli topoisomerase IV (0.15 and 0.6 U) was carried out as detailed in the Materials and Methods section using HMGB1 or mutant (1 and 4 μM, left to right). The DNA samples in (C–E) were separated on 0.8% agarose gels without (C) or with (D–E) ethidium bromide in the course of electrophoresis as detailed in the Materials and Methods section. The gels are presented as negatives. WT, wild-type HMGB1; Mut, triple-mutant (F38A/F103A/I122A) of HMGB1.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Activity Assay, Mutagenesis, Electrophoresis, Staining, Introduce, Plasmid Preparation, Incubation

    HMGB1 stimulates decatenation of kinetoplast DNA and relaxation of supercoiled DNA by topoisomerase IIα. ( A ) Decatenation of kinetoplast DNA (kDNA) by topo IIα is stimulated by HMGB1. kDNA (0.2 μg) was incubated with increasing concentrations of topo IIα (as indicated) in the absence or presence of HMGB1 (1 μM). ( B ) Domain structure of HMGB1, HMGB1-ΔC, HMGB1 lacking the acidic C-tail (depicted as oval); A, domain A; B, domain B. ( C ) HMGB1 stimulates decatenation of kDNA by topo IIα via the HMG-box domains. kDNA (0.2 μg) was incubated with topo IIα (2 nM) in the presence or absence of increasing amounts of HMGB1, HMGB1-ΔC, domain A or domain B (as indicated). ΔC, HMGB1-ΔC. ( D ) HMGB1 stimulates relaxation of negatively supercoiled DNA by HMGB1. Relaxation of negatively supercoiled pBR322 plasmid DNA (∼8 nM) by different concentrations of topo IIα (as indicated) in the presence or absence of recombinant HMGB1 (1 μM), 37°C for 45 min. Decatenation and relaxation assays were carried out in the absence of PEG. Deproteinized DNA samples were resolved on 1% agarose gels containing ethidium bromide (A and C) or without ethidium bromide (panel D). The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. oc, nicked closed-circular DNA minicircle; rel, relaxed closed-circular DNA minicircle.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 stimulates decatenation of kinetoplast DNA and relaxation of supercoiled DNA by topoisomerase IIα. ( A ) Decatenation of kinetoplast DNA (kDNA) by topo IIα is stimulated by HMGB1. kDNA (0.2 μg) was incubated with increasing concentrations of topo IIα (as indicated) in the absence or presence of HMGB1 (1 μM). ( B ) Domain structure of HMGB1, HMGB1-ΔC, HMGB1 lacking the acidic C-tail (depicted as oval); A, domain A; B, domain B. ( C ) HMGB1 stimulates decatenation of kDNA by topo IIα via the HMG-box domains. kDNA (0.2 μg) was incubated with topo IIα (2 nM) in the presence or absence of increasing amounts of HMGB1, HMGB1-ΔC, domain A or domain B (as indicated). ΔC, HMGB1-ΔC. ( D ) HMGB1 stimulates relaxation of negatively supercoiled DNA by HMGB1. Relaxation of negatively supercoiled pBR322 plasmid DNA (∼8 nM) by different concentrations of topo IIα (as indicated) in the presence or absence of recombinant HMGB1 (1 μM), 37°C for 45 min. Decatenation and relaxation assays were carried out in the absence of PEG. Deproteinized DNA samples were resolved on 1% agarose gels containing ethidium bromide (A and C) or without ethidium bromide (panel D). The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. oc, nicked closed-circular DNA minicircle; rel, relaxed closed-circular DNA minicircle.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Incubation, Plasmid Preparation, Recombinant

    HMGB1 stimulates DNA cleavage activity of topoisomerase IIα. ( A ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the absence of etoposide. DNA cleavage reactions contained negatively supercoiled plasmid pBR322 (∼4 nM), human topo IIα (∼8 nM) and increasing concentrations of HMGB1 (1, 2 and 3 μM, left to right). ATP or a non-hydrolyzable ATP analog (AMP-PNP) were at 1 mM. Arrowhead indicates position of linearized DNA. ( B ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the presence of etoposide. The molar concentrations of topo IIα and plasmid pBR322 were identical as in panel A. HMGB1 protein was at 1 μM and etoposide at 50 μM. ( C ) Effect of HMGB1 on DNA cleavage by topo IIα in the presence of increasing concentrations of etoposide. Negatively supercoiled plasmid DNA (4 nM ) was pre-incubated on ice either with buffer (control) or HMGB1 (1 μM) in the presence of 0–100 μM etoposide. Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation was carried out at 37°C for 15 min. The cleavage complexes in A–C reactions were trapped by 1% SDS, followed by digestion with proteinase K as detailed in Materials and Methods section. Deproteinized DNA samples were resolved on 1% agarose gels and subsequently stained with ethidium bromide. FIII, Hin dIII-digested plasmid pBR322 (last right lanes in A and B) indicates mobility of linearized plasmid DNA. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. The gels are presented as negatives. The percentage of cleaved plasmid DNA was quantified from three independent cleavage experiments, each in duplicates, by ImageQuant TL (GE Healthcare). The average SD for the data was

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 stimulates DNA cleavage activity of topoisomerase IIα. ( A ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the absence of etoposide. DNA cleavage reactions contained negatively supercoiled plasmid pBR322 (∼4 nM), human topo IIα (∼8 nM) and increasing concentrations of HMGB1 (1, 2 and 3 μM, left to right). ATP or a non-hydrolyzable ATP analog (AMP-PNP) were at 1 mM. Arrowhead indicates position of linearized DNA. ( B ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the presence of etoposide. The molar concentrations of topo IIα and plasmid pBR322 were identical as in panel A. HMGB1 protein was at 1 μM and etoposide at 50 μM. ( C ) Effect of HMGB1 on DNA cleavage by topo IIα in the presence of increasing concentrations of etoposide. Negatively supercoiled plasmid DNA (4 nM ) was pre-incubated on ice either with buffer (control) or HMGB1 (1 μM) in the presence of 0–100 μM etoposide. Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation was carried out at 37°C for 15 min. The cleavage complexes in A–C reactions were trapped by 1% SDS, followed by digestion with proteinase K as detailed in Materials and Methods section. Deproteinized DNA samples were resolved on 1% agarose gels and subsequently stained with ethidium bromide. FIII, Hin dIII-digested plasmid pBR322 (last right lanes in A and B) indicates mobility of linearized plasmid DNA. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. The gels are presented as negatives. The percentage of cleaved plasmid DNA was quantified from three independent cleavage experiments, each in duplicates, by ImageQuant TL (GE Healthcare). The average SD for the data was

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Staining

    Electrophoresis of DNA plasmid pBR322 incubated in 5 mM Tris/HCl buffer for 20 h at 37°C with cisplatin, CQDP, complex 1 and complex 6 . Lanes 1 and 13: molecular marker; lane 2: DNA pBR322; lane 3: DNA treated with 0.5 eq. of cisplatin; lanes

    Journal: Journal of inorganic biochemistry

    Article Title: Arene-Ru(II)-Chloroquine Complexes Interact With DNA, Induce Apoptosis on Human Lymphoid Cell Lines and Display Low Toxicity to Normal Mammalian Cells

    doi: 10.1016/j.jinorgbio.2010.05.002

    Figure Lengend Snippet: Electrophoresis of DNA plasmid pBR322 incubated in 5 mM Tris/HCl buffer for 20 h at 37°C with cisplatin, CQDP, complex 1 and complex 6 . Lanes 1 and 13: molecular marker; lane 2: DNA pBR322; lane 3: DNA treated with 0.5 eq. of cisplatin; lanes

    Article Snippet: Calf Thymus (CT) DNA, pBR322 plasmid DNA, buffers and solvents were purchased from Sigma-Aldrich.

    Techniques: Electrophoresis, Plasmid Preparation, Incubation, Marker

    The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the pBR322-Dunlop or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.

    Journal: Virology

    Article Title: A System for the Analysis of BKV Non-coding Control Regions: Application to Clinical Isolates from an HIV/AIDS Patient

    doi: 10.1016/j.virol.2010.08.032

    Figure Lengend Snippet: The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the pBR322-Dunlop or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.

    Article Snippet: The pBR322-Dunlop plasmid (ATCC #45025, pBKV) was received from Peter Howley ( ).

    Techniques: Transfection, Molecular Weight, Southern Blot, Construct, Marker