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  • 98
    New England Biolabs saci
    Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with <t>PvuII,</t> MfeI, and <t>SacI</t> to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.
    Saci, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 2245 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher saci fastdigest
    Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with <t>PvuII,</t> MfeI, and <t>SacI</t> to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.
    Saci Fastdigest, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore saci
    Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with <t>PvuII,</t> MfeI, and <t>SacI</t> to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.
    Saci, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher saci
    (I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [ γ - 32 P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short <t>DNA</t> fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two <t>SacI</t> recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗ U: uracil; ∗∗ AP: apurinic/apyrimidinic.
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    95
    New England Biolabs saci hf
    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with <t>SacI</t> and <t>SpeI</t> restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.
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    Image Search Results


    Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with PvuII, MfeI, and SacI to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.

    Journal: The Plant Cell

    Article Title: The Transcriptional Enhancer of the Pea Plastocyanin Gene Associates with the Nuclear Matrix and Regulates Gene Expression through Histone Acetylation

    doi: 10.1105/tpc.011825

    Figure Lengend Snippet: Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with PvuII, MfeI, and SacI to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.

    Article Snippet: After six washes with buffer D (20 mM Tris-HCl, pH 8.0, 70 mM NaCl, 20 mM KCl, 10 mM MgCl2 , 0.125 mM spermidine, 0.05 mM spermine, and 0.1% digitonin), the matrices were suspended in 500 μL of buffer D, and 200 units of PvuII, MfeI, and SacI (New England Biolabs, Beverly, MA) were added.

    Techniques: Isolation, Centrifugation, Polymerase Chain Reaction, Amplification

    Restriction analysis of pKBuS13 separation on 0.8% agarose gel electrophoresis of pKBuS13 extracted from the E. coli JM101 recipient and digested with BamHI (lane B), HindIII (lane H), SacI (lane S), and PstI (lane P). Lane M, GeneRuler 1-kb DNA ladder (Thermo Scientific).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

    doi: 10.1128/AAC.04543-14

    Figure Lengend Snippet: Restriction analysis of pKBuS13 separation on 0.8% agarose gel electrophoresis of pKBuS13 extracted from the E. coli JM101 recipient and digested with BamHI (lane B), HindIII (lane H), SacI (lane S), and PstI (lane P). Lane M, GeneRuler 1-kb DNA ladder (Thermo Scientific).

    Article Snippet: Plasmid restriction analysis was carried on with BamHI, HindIII, PstI, and SacI restriction enzymes according to the manufacturer's instructions (New England BioLabs, Mississauga, Ontario, Canada) followed by separation on 0.8% agarose gel.

    Techniques: Agarose Gel Electrophoresis

    (I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [ γ - 32 P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗ U: uracil; ∗∗ AP: apurinic/apyrimidinic.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Efficiency of Base Excision Repair of Oxidative DNA Damage and Its Impact on the Risk of Colorectal Cancer in the Polish Population

    doi: 10.1155/2016/3125989

    Figure Lengend Snippet: (I) A uracil-containing oligonucleotide was subjected to action of polynucleotide kinase to attach a radioactive phosphate group from [ γ - 32 P] ATP. It was hybridized with a second oligonucleotide whose sequence was adjusted to obtain sticky ends referring to XhoI and XbaI digestion site. (II) The short DNA fragment prepared in stage I was cloned into a pBluescriptII plasmid. (III) Uracil-DNA glycosylase was utilized to remove uracil and, as consequence, create a single gap in DNA to act as a synthetic lesion. (IV) A plasmid with single AP site constituted a substrate for the protein extract in 90-minute repair incubation. (V) Two SacI recognition sites of the pBluescriptII plasmid were used to excise 450 pb-long fragment covering the lesion site and radioactive label for analysis on 8% urea/acrylamide gel. (VI) Interpretation of outcomes was based on detection of two bands. The full-length 450 pb fragment reflects restored DNA fraction, whereas presence of short 180 pb fraction indicates the amount of unrepaired DNA. ∗ U: uracil; ∗∗ AP: apurinic/apyrimidinic.

    Article Snippet: A 450 pb length DNA fragment was excised from plasmid by 1 U of SacI enzyme (ThermoScientific, Rochester, USA) incubated at 37°C for 1 h. To allow the repaired and unrepaired fractions to be differentiated, the remaining unrepaired AP site was treated with 1 U of AP-recognizing endonuclease IV for 1 h. All samples were run on 8% urea-containing polyacrylamide gel for 3 h in 120 V. The accurate electrophoresis was preceded by 1 h preelectrophoresis with loading buffer.

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Activated Clotting Time Assay, Incubation, Acrylamide Gel Assay

    Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Mobilizable Plasmids for Tunable Gene Expression in Francisella novicida

    doi: 10.3389/fcimb.2018.00284

    Figure Lengend Snippet: Plasmid stability. GFP expression was induced with 500 ng/ml of ATc. ON culture of day 0 was supplemented with 15 μg/ml kanamycin, while ON cultures of days 1–4 were supplemented with 50 μg/ml ampicillin. (A) GFP expression in F. novicida U112 pFNMB1 msfgfp ON cultures of day 0 and day 4. (B) GFP expression in F. novicida U112 pFNMB2 msfgfp ON cultures of day 0 and day 4. (A,B) Images are a merge of phase contrast and GFP channel. 26 × 39 μm fields of view are shown. Scale bar represent 5 μm. Representative replicates are shown. Three independent experiments were performed. (C) Survival assay performed with ON cultures of F. novicida U112 pFNMB1 msfgfp and F. novicida U112 pFNMB2 msfgfp plated on ampicillin and ampicillin/kanamycin plates. Three independent experiments were performed. (D) Digestion of pFNMB1 msfgfp with SacI and SpeI restriction enzymes. (E) Digestion of pFNMB2 msfgfp with SacI and SpeI restriction enzymes. (D,E) Plasmids were passaged in F. novicida for 4 days before being transformed into E. coli . Controls were isolated directly from E. coli . Representative replicates are shown. Three independent experiments were performed.

    Article Snippet: The next day, colonies were grown in liquid LB supplemented with 50 μg/ml kanamycin, plasmid DNA was isolated and 250 ng of each plasmid was digested with SacI-HF and SpeI restriction enzymes (New England BioLabs) for 1 h. As control, both plasmids were additionally isolated from E. coli directly, without passaging in F. novicida , and digested identically.

    Techniques: Plasmid Preparation, Expressing, Clonogenic Cell Survival Assay, Transformation Assay, Isolation

    Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Journal: Nucleic Acids Research

    Article Title: Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice

    doi: 10.1093/nar/gky456

    Figure Lengend Snippet: Heart Sod2 knockout mice show normal mtDNA topology and no decrease in mtDNA copy number. ( A ) Representative phosphorimager exposure of mtDNA topology analysis of total DNA from heart tissue from 10-week old Sod2 loxP x Ckmm cre mice. MtDNA is visualized using radioactive probes towards mtDNA. Control DNA was treated with various enzymes to reveal the different topologies of mtDNA. SacI cuts both strands of mtDNA once (linear), Nt. BbvCI cuts only one strand of mtDNA (nicked), TopoI relaxes the mtDNA (looser coiling), Gyrase creates coiling to mtDNA (compacted supercoiled DNA). Experimental samples are untreated. First gel does not have ethidium bromide (EtBr), second gel has the same samples and EtBr in the gel to compact the closed circle DNA into a quantifiable band. Phosphorimager images are filtered with averaging to reduce noise. Quantifications were made from the original images. ( B ) Quantification of the proportion of closed circle form of mtDNA per total mtDNA. Quantification is done from phosphorimager exposure of the topology gels. White circles indicate samples from controls (pp, n = 11, 9–10 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp,cre, n = 12, 10-week old). ( C ) Relative mtDNA copy number in heart of Sod2 loxP x Ckmm cre mice as assessed with qPCR. MtDNA levels were analyzed with a CytB probe and nuclear DNA with a 18S probe. White circles indicate samples from controls (pp, n = 12, 10–12 week old) and gray circles indicate samples from Sod2 loxP x Ckmm cre mice (pp, cre, n = 11, 10–12 week old). Horizontal lines represent means, error bars represent SD, * P

    Article Snippet: The DNA was digested overnight with SacI-HF restriction enzyme (New England Biolabs) and ethanol precipitated.

    Techniques: Knock-Out, Mouse Assay, Real-time Polymerase Chain Reaction