dh5α Thermo Fisher Search Results


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  • 96
    Thermo Fisher max efficiency dh5alpha
    In vivo DNA assembly and cloning in E . coli <t>DH5α.</t> ( A ) E . coli DH5α-mediated DNA assembly involves only two basic steps: 1) preparation of fragments with homologous ends and 2) introduction of the fragments into competent cells. This approach minimizes the time and reagents required for DNA assembly in comparison to other common methods, which contain a separate assembly step before the introduction of the constructed plasmid into a recombination-impaired cloning strain such as DH5α ( B-D ). Assembly is typically carried out either with added enzymes in vitro ( B ), or in vivo , using as a host S . cerevisiae ( C ) or specialized E . coli strains expressing the λ Red or RecET phage-based systems ( D ).
    Max Efficiency Dh5alpha, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    92
    Thermo Fisher strain dh5 α thermo fisher scientific
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    80
    Thermo Fisher dh5α f
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    90
    Thermo Fisher competent dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Competent Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher ec dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    92
    Thermo Fisher electromax dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Electromax Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher dh5α t1
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    90
    Thermo Fisher dh5α e
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    88
    Thermo Fisher host dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    85
    Thermo Fisher dh5α mcr
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Dh5α Mcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher subcloning efficiency dh5alpha cells
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Subcloning Efficiency Dh5alpha Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher electromax dh5α e
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Electromax Dh5α E, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher dh5α bacteria
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
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    89
    Thermo Fisher strain dh5α
    E. coli <t>DH5α</t> cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.
    Strain Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vivo DNA assembly and cloning in E . coli DH5α. ( A ) E . coli DH5α-mediated DNA assembly involves only two basic steps: 1) preparation of fragments with homologous ends and 2) introduction of the fragments into competent cells. This approach minimizes the time and reagents required for DNA assembly in comparison to other common methods, which contain a separate assembly step before the introduction of the constructed plasmid into a recombination-impaired cloning strain such as DH5α ( B-D ). Assembly is typically carried out either with added enzymes in vitro ( B ), or in vivo , using as a host S . cerevisiae ( C ) or specialized E . coli strains expressing the λ Red or RecET phage-based systems ( D ).

    Journal: PLoS ONE

    Article Title: Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

    doi: 10.1371/journal.pone.0137466

    Figure Lengend Snippet: In vivo DNA assembly and cloning in E . coli DH5α. ( A ) E . coli DH5α-mediated DNA assembly involves only two basic steps: 1) preparation of fragments with homologous ends and 2) introduction of the fragments into competent cells. This approach minimizes the time and reagents required for DNA assembly in comparison to other common methods, which contain a separate assembly step before the introduction of the constructed plasmid into a recombination-impaired cloning strain such as DH5α ( B-D ). Assembly is typically carried out either with added enzymes in vitro ( B ), or in vivo , using as a host S . cerevisiae ( C ) or specialized E . coli strains expressing the λ Red or RecET phage-based systems ( D ).

    Article Snippet: Bacterial strains The following commercial products were used: Max Efficiency DH5α (Life Technologies, Carlsbad, CA; chemically competent, ~109 colony-forming unit or CFU / μg pUC19), High Efficiency NEB 5-alpha (New England Biolabs, Ipswich, MA; chemically competent, CFU ~109 /μg pUC19), and NEB 5-alpha Electrocompetent E . coli (New England Biolabs, CFU ~1010 /μg pUC19).

    Techniques: In Vivo, Clone Assay, Construct, Plasmid Preparation, In Vitro, Expressing

    E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

    Journal: Nucleic Acids Research

    Article Title: Functional replacement of the endogenous tyrosyl-tRNA synthetase-tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

    doi: 10.1093/nar/gkq080

    Figure Lengend Snippet: E. coli DH5α cells, an E. coli K-12 strain, expressing the wild-type E. coli TyrRS from plasmid pEcYS (filled circles) and those expressing iodoTyrRS- ec from plasmid pEcIYS (open circles) were grown in the presence of 3-bromo- l -tyrosine (0.5 mg/ml) ( A ) and 3-azido- l -tyrosine (0.3 mg/ml) ( B ). FT3 cells expressing iodoTyrRS- ec were grown in the absence (filled circles) and presence (open circles) of 3-bromo- l -tyrosine ( C ). The growth was monitored upon the addition of the non-natural amino acid to the growth media.

    Article Snippet: Strains, growth conditions, growth rate, reagents and non-natural amino acids Escherichia coli strains TOP10, BL21(DE3) and DH5α were purchased from Invitrogen, Novagen and Toyobo (Tokyo, Japan), respectively.

    Techniques: Expressing, Plasmid Preparation

    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Journal: Scientific Reports

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    doi: 10.1038/s41598-018-30792-0

    Figure Lengend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Article Snippet: Bacterial strains E . coli DH5α purchased from Life Technologies, genotype F− Φ80lac ZΔM15 Δ(lac ZYA-arg F) U169 rec A1 end A1 hsd R17 (rK−, mK+) pho A sup E44 λ− thi -1 gyr A96 rel A1 λ- was used for plasmids clonning and long term storage.

    Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    NID-extracted DNA of both high and low copy number plasmids perform well in common downstream applications. ( A ) Restriction endonuclease digestion of the high copy number pLTM330 plasmid containing 2 SacI sites separated by approximately 400 bp. extracted by either alkali or NIDs. XL1-Blue cells harboring pLTM330 in 1.5 ml LB cultures were used for isolation. After alcohol precipitation, alkali DNA pellets were rinsed with 0.5 ml ethanol and dissolved in 40 µl TE buffer, but NID DNA pellets were directly dissolved in 40 µl TE buffer. 9 µl aliquots of native DNA of two independently isolated samples were loaded in lanes 1, 2 (alkali, A) and lanes 3, 4 (NID, N). Densitometry analysis is reported for lanes 1–4. 9 µl DNA was also used for every DNA restriction and ligation reaction in 11 µl total volume. Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol. Arrow indicates “irreversibly denatured” DNA [1] , [25] , [29] . Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5–12. DNA restriction reactions were carried out in NEB1 buffer. ( B ) Plasmid samples digested using 2u SacI for 1 hour (lanes 1,4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15°C for 30 minutes (lanes 2,5). Ligation products were then re-digested using 2u SacI for 1 hour (lanes 3,6). DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Arrowhead indicates 400 bp band. B = cut sample. L = ligated sample. D = re-cut sample. Lane 2 shows the intermediate ligation products of the 400 bp DNA fragment in lane 1. ( C ) DH5α cells harboring the low copy number plasmid pEL04, containing 2 KpnI sites separated by approximately 1.5 Kb. NID plasmid isolation was performed as reported in Materials and Methods , except that lysozyme concentration in the extraction buffer was 50 µg/ml. 9 µl DNA in 11 µl total reaction volume were used for digestions. Lane 1: native DNA (N). Lane 2: DNA digestion by 1.5u KpnI. Lane 3: 3u KpnI. Lane 4: 5u KpnI. The incubation times were 1 hour. Lane 5: 1 Kb Plus DNA Ladder (Invitrogen). Densitometry analysis of the expected 1.5 Kb digestion product is reported in lanes 2–4.

    Journal: PLoS ONE

    Article Title: A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

    doi: 10.1371/journal.pone.0023457

    Figure Lengend Snippet: NID-extracted DNA of both high and low copy number plasmids perform well in common downstream applications. ( A ) Restriction endonuclease digestion of the high copy number pLTM330 plasmid containing 2 SacI sites separated by approximately 400 bp. extracted by either alkali or NIDs. XL1-Blue cells harboring pLTM330 in 1.5 ml LB cultures were used for isolation. After alcohol precipitation, alkali DNA pellets were rinsed with 0.5 ml ethanol and dissolved in 40 µl TE buffer, but NID DNA pellets were directly dissolved in 40 µl TE buffer. 9 µl aliquots of native DNA of two independently isolated samples were loaded in lanes 1, 2 (alkali, A) and lanes 3, 4 (NID, N). Densitometry analysis is reported for lanes 1–4. 9 µl DNA was also used for every DNA restriction and ligation reaction in 11 µl total volume. Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol. Arrow indicates “irreversibly denatured” DNA [1] , [25] , [29] . Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5–12. DNA restriction reactions were carried out in NEB1 buffer. ( B ) Plasmid samples digested using 2u SacI for 1 hour (lanes 1,4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15°C for 30 minutes (lanes 2,5). Ligation products were then re-digested using 2u SacI for 1 hour (lanes 3,6). DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Arrowhead indicates 400 bp band. B = cut sample. L = ligated sample. D = re-cut sample. Lane 2 shows the intermediate ligation products of the 400 bp DNA fragment in lane 1. ( C ) DH5α cells harboring the low copy number plasmid pEL04, containing 2 KpnI sites separated by approximately 1.5 Kb. NID plasmid isolation was performed as reported in Materials and Methods , except that lysozyme concentration in the extraction buffer was 50 µg/ml. 9 µl DNA in 11 µl total reaction volume were used for digestions. Lane 1: native DNA (N). Lane 2: DNA digestion by 1.5u KpnI. Lane 3: 3u KpnI. Lane 4: 5u KpnI. The incubation times were 1 hour. Lane 5: 1 Kb Plus DNA Ladder (Invitrogen). Densitometry analysis of the expected 1.5 Kb digestion product is reported in lanes 2–4.

    Article Snippet: DH5α cells were supplied by Invitrogen (Carlsbad, CA).

    Techniques: Low Copy Number, Plasmid Preparation, Isolation, Ligation, Incubation, Concentration Assay

    Elevated temperatures and osmolytes increase the efficiency of plasmid DNA extraction by NIDs. Extractions of either pUC19 (lanes 1–8 and 10–15) or pCYPAC3 plasmids (lane 9) were carried out. Transformed DH5α cells were grown in 1.5 ml LB cultures and resuspended in 150 µl 50 mM Tris pH 8, 10 mM EDTA with or without co-solutes. 500 µg/ml lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 12–15. Extracts were cleared by centrifugation, and precipitated with 150 µl isopropanol. DNA pellets were dissolved in 40 µl TE buffer, 40 µg/ml RNase A. 10 µl aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1–9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10–15, the included co-solute is indicated. Lane 1: 0.5% IGEPAL CA-630. Lane 2: 0.5% TX-100. Lane 3: 0.5% IGEPAL CA-720. Lane 4: 0.5% Tween-80. Lane 5: 0.5% Tween-20. Lane 6: 2% IGEPAL CA-720. Lane 7: 4% IGEPAL CA-720. Lane 8: 0.5% Tween 80. Lane 9: 0.5% Tween 80. Lane 10: 0.5% Tween 20/0.5 M KCl. Lane 11: 0.5% Tween 20/22.5% sucrose. Lane 12: 0.5% IGEPAL CA-630/0.5 M NH 4 Cl. Lane 13: 0.5% TX-100/0.5 M NH 4 Cl. Lane 14: 0.5% TX-100/0.5 M NaCl. Lane 15: 0.5% TX-100/0.5 M NaAc.

    Journal: PLoS ONE

    Article Title: A One-Step Miniprep for the Isolation of Plasmid DNA and Lambda Phage Particles

    doi: 10.1371/journal.pone.0023457

    Figure Lengend Snippet: Elevated temperatures and osmolytes increase the efficiency of plasmid DNA extraction by NIDs. Extractions of either pUC19 (lanes 1–8 and 10–15) or pCYPAC3 plasmids (lane 9) were carried out. Transformed DH5α cells were grown in 1.5 ml LB cultures and resuspended in 150 µl 50 mM Tris pH 8, 10 mM EDTA with or without co-solutes. 500 µg/ml lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCl in all extracts, except the ones loaded in lanes 10 and 12–15. Extracts were cleared by centrifugation, and precipitated with 150 µl isopropanol. DNA pellets were dissolved in 40 µl TE buffer, 40 µg/ml RNase A. 10 µl aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1–9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10–15, the included co-solute is indicated. Lane 1: 0.5% IGEPAL CA-630. Lane 2: 0.5% TX-100. Lane 3: 0.5% IGEPAL CA-720. Lane 4: 0.5% Tween-80. Lane 5: 0.5% Tween-20. Lane 6: 2% IGEPAL CA-720. Lane 7: 4% IGEPAL CA-720. Lane 8: 0.5% Tween 80. Lane 9: 0.5% Tween 80. Lane 10: 0.5% Tween 20/0.5 M KCl. Lane 11: 0.5% Tween 20/22.5% sucrose. Lane 12: 0.5% IGEPAL CA-630/0.5 M NH 4 Cl. Lane 13: 0.5% TX-100/0.5 M NH 4 Cl. Lane 14: 0.5% TX-100/0.5 M NaCl. Lane 15: 0.5% TX-100/0.5 M NaAc.

    Article Snippet: DH5α cells were supplied by Invitrogen (Carlsbad, CA).

    Techniques: Plasmid Preparation, DNA Extraction, Transformation Assay, Incubation, Centrifugation