Structured Review

GE Healthcare puc19
Hemolysis of human red blood cells by recombinant E. coli containing L. pneumophila plaB . (A) Human blood agar was inoculated with recombinant E. coli harboring different plasmids and then incubated for 48 h at 37°C. Plasmids contained the following inserts: pKH190 and pKH192, intact plaB ; pKH194, disrupted plaB ; <t>pUC19,</t> empty vector; p hlyCABD , hemolysin-encoding operon of E. coli 536 in pUC18 (compare Fig. ). (B) Recombinant E. coli containing either plain pBCKS+ or pBCKS+ with L. pneumophila plaB (pKH192) was grown to the logarithmic-growth phase and subsequently treated with IPTG. Afterwards, human red blood cells were incubated with the bacteria for 20 h and then quantitatively assessed for hemolysis. Results are means and standard deviations from triplicate cultures and are representative of three independent experiments.
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Images

1) Product Images from "Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity "

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity

Journal:

doi: 10.1128/IAI.72.5.2648-2658.2004

Hemolysis of human red blood cells by recombinant E. coli containing L. pneumophila plaB . (A) Human blood agar was inoculated with recombinant E. coli harboring different plasmids and then incubated for 48 h at 37°C. Plasmids contained the following inserts: pKH190 and pKH192, intact plaB ; pKH194, disrupted plaB ; pUC19, empty vector; p hlyCABD , hemolysin-encoding operon of E. coli 536 in pUC18 (compare Fig. ). (B) Recombinant E. coli containing either plain pBCKS+ or pBCKS+ with L. pneumophila plaB (pKH192) was grown to the logarithmic-growth phase and subsequently treated with IPTG. Afterwards, human red blood cells were incubated with the bacteria for 20 h and then quantitatively assessed for hemolysis. Results are means and standard deviations from triplicate cultures and are representative of three independent experiments.
Figure Legend Snippet: Hemolysis of human red blood cells by recombinant E. coli containing L. pneumophila plaB . (A) Human blood agar was inoculated with recombinant E. coli harboring different plasmids and then incubated for 48 h at 37°C. Plasmids contained the following inserts: pKH190 and pKH192, intact plaB ; pKH194, disrupted plaB ; pUC19, empty vector; p hlyCABD , hemolysin-encoding operon of E. coli 536 in pUC18 (compare Fig. ). (B) Recombinant E. coli containing either plain pBCKS+ or pBCKS+ with L. pneumophila plaB (pKH192) was grown to the logarithmic-growth phase and subsequently treated with IPTG. Afterwards, human red blood cells were incubated with the bacteria for 20 h and then quantitatively assessed for hemolysis. Results are means and standard deviations from triplicate cultures and are representative of three independent experiments.

Techniques Used: Recombinant, Incubation, Plasmid Preparation

2) Product Images from "The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility"

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049551

Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
Figure Legend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

Techniques Used: Incubation, Produced, Marker

3) Product Images from "Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria"

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria

Journal:

doi: 10.1128/JB.00824-07

Subcloning of the open reading frame associated with adsorption of φ24B to MRL1. Construct pUCR1 was one of the four original clones that complemented the φ24B phage adsorption defect. All four clones shared the two open reading frames present in pUCR1; the solid box represents yaeT , and the striped box represents skp . When fragments from pUCR1 were subcloned into pUC19 or pUC18 (indicated by the vector promoter direction: leftward, pUC19; rightward, pUC18), only the complete yaeT gene found in pUCR1 and pUCR1D complemented the MRL1 adsorption defect. Restriction sites are as follows: X, XhoI; H, HincII; K, KpnI; P, PstI.
Figure Legend Snippet: Subcloning of the open reading frame associated with adsorption of φ24B to MRL1. Construct pUCR1 was one of the four original clones that complemented the φ24B phage adsorption defect. All four clones shared the two open reading frames present in pUCR1; the solid box represents yaeT , and the striped box represents skp . When fragments from pUCR1 were subcloned into pUC19 or pUC18 (indicated by the vector promoter direction: leftward, pUC19; rightward, pUC18), only the complete yaeT gene found in pUCR1 and pUCR1D complemented the MRL1 adsorption defect. Restriction sites are as follows: X, XhoI; H, HincII; K, KpnI; P, PstI.

Techniques Used: Subcloning, Adsorption, Construct, Clone Assay, Plasmid Preparation

4) Product Images from "The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility"

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

Journal: PLoS ONE

doi: 10.1371/journal.pone.0049551

Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
Figure Legend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

Techniques Used: Incubation, Produced, Marker

Related Articles

Clone Assay:

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The erm cassette was amplified from plasmid pVA2198 ( ). .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. Ligated fragments were introduced into P. gingivalis ATCC 33277 by electroporation.

Article Title: Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family
Article Snippet: Strains DY330 and MG1655 were used for the construction of knockout strains. .. Plasmids pBAD/Myc -His B (Invitrogen), pACYC184, pUC19 and pKK232-8 (Amersham Pharmacia) were used as cloning vectors. .. Plasmid pSS6 ( ) was used for construction of an IPTG-inducible RNA expression vector.

Article Title: A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions
Article Snippet: Other common E. coli expression strains, including C41(DE3), C43(DE3), and BL21(DE3), were also tested ( ). .. Plasmids used were pUC19 ( ) for cloning and pTrc99A (Amersham Biosciences) for overexpression of A1 Ao -ATP synthase. .. To overproduce the A1 Ao -ATP synthase, transformants of E. coli DK8 Δ atp were routinely grown at 37 °C with shaking at 200 rpm in 2× YT medium ( ) containing 2 g/liter glucose and 100 μg of ampicillin/ml.

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: The seed-specific promoter together with the relative 5′ UTR and transit peptide sequence was amplified from soybean DNA with primers inserting an XbaI and BamHI site (forward primer: TCT AGA GTT TTC AAA TTT GAA TTT TAA TGT GTG TTG and reverse primer: GGA TCC CAC CTT AAG GAG GTT GCA ACG AGC GTG GCA). .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene. .. The engineered plasmids were introduced in Agrobacterium tumefaciens strain EHA105 by electroporation.

Article Title: Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana
Article Snippet: Cesium chloride-purified BAC DNA was sheared by nebulization ( ). .. After end-filling, DNA fragments were size fractionated and cloned into the Sma I site of pUC18 or Hin cII site of pUC19 (Amersham Pharmacia Biotech). .. Clones were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing ready Reaction kit with FS AmpliTaq DNA polymerase (PE Applied Biosystems) and analyzed on ABI 377 (PE Applied Biosystems) sequencing gels.

Amplification:

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility
Article Snippet: A 1075 bp DNA fragment comprising the predicted I-Uma I target site was amplified from genomic DNA of U. maydis strain 521 (F type) using the primer combination 5′-GGAATTC CATATG CTCCTCGCCGAATACGAGAGG-3′ )/5′-GGAATTC CATATG TCCCAGTCAAACTGACCACC-3′ (Nde I sites underlined) and inserted into the Nde I site of pSL1180 (GE Healthcare, Darmstadt, Germany) to yield pSL521. .. Cleaved fragments were ligated in the presence of T4 ligase (NEB) into the compatible sites of pUC19 (GE Healthcare).

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The erm cassette was amplified from plasmid pVA2198 ( ). .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan).

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: Paragraph title: RNA Extraction, cDNA Amplification, and Cloning ... Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Construct:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: An expression library of L. pneumophila Corby was constructed as described previously ( ). .. The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ).

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: Porphyromonas gingivalis deletion mutants lacking PGN_1171, PGN_0725, PGN_1341, and/or PGN_1888 gene were constructed by replacing each gene with ermF and ermB (erm cassette), as previously described ( ). .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan).

Article Title: Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication
Article Snippet: The plasmid pORI(wt) has been described ( ). pUC19 was purchased from Amersham Pharmacia. .. The plasmid pORI(wt) has been described ( ). pUC19 was purchased from Amersham Pharmacia.

Incubation:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. Transformants were isolated on LB agar containing 100 μg ml−1 ampicillin and then used to inoculate LB-Ca.

Expressing:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: An expression library of L. pneumophila Corby was constructed as described previously ( ). .. The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ).

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: The level of phage adsorption was determined as follows: percentage of phage adsorption = [(PFU from LB-Ca control − PFU from sample infection)/PFU from wild-type MC1061 infection] × 100. .. A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. This library was transformed into MC1061-MRL1.

Article Title: A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions
Article Snippet: Other common E. coli expression strains, including C41(DE3), C43(DE3), and BL21(DE3), were also tested ( ). .. Plasmids used were pUC19 ( ) for cloning and pTrc99A (Amersham Biosciences) for overexpression of A1 Ao -ATP synthase.

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: An EcoRV site was inserted respectively in the forward and in the reverse primer to facilitate subsequent cDNA cloning in the plant expression vector. .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Over Expression:

Article Title: A1Ao-ATP Synthase of Methanobrevibacter ruminantium Couples Sodium Ions for ATP Synthesis under Physiological Conditions
Article Snippet: Other common E. coli expression strains, including C41(DE3), C43(DE3), and BL21(DE3), were also tested ( ). .. Plasmids used were pUC19 ( ) for cloning and pTrc99A (Amersham Biosciences) for overexpression of A1 Ao -ATP synthase. .. To overproduce the A1 Ao -ATP synthase, transformants of E. coli DK8 Δ atp were routinely grown at 37 °C with shaking at 200 rpm in 2× YT medium ( ) containing 2 g/liter glucose and 100 μg of ampicillin/ml.

Countercurrent Chromatography:

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: Amplification of the GAA coding sequence was performed by combining the reverse primer with a second forward primer (GAT ATC TGC ACA CCC CGG CCG TCC CAG) matching the 5′ terminus of the cDNA sequence (GenBank acc. .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Electroporation:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ). .. PCR was carried out using a TRIO-Thermoblock or a T-Gradient thermocycler (Biometra, Göttingen, Germany) and AmpliTaq polymerase (Perkin-Elmer, Weiterstadt, Germany) or Taq DNA polymerase (New England Biolabs, Frankfurt am Main, Germany).

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. To create double mutants, tetracycline resistance gene tetQ , amplified from plasmid pT-COW , was used as an additional selectable marker.

Ligation:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: The level of phage adsorption was determined as follows: percentage of phage adsorption = [(PFU from LB-Ca control − PFU from sample infection)/PFU from wild-type MC1061 infection] × 100. .. A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. This library was transformed into MC1061-MRL1.

Infection:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. Transformants were isolated on LB agar containing 100 μg ml−1 ampicillin and then used to inoculate LB-Ca.

Sequencing:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: Paragraph title: DNA techniques and sequence analysis. ... The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ).

Article Title: Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses
Article Snippet: E. coli strain hosts XL1-Blue and DH10B were grown in LB or Terrific broth . .. The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega). .. DNA inserts were sequenced using the DYEnamic ET kit (GE HealthCare) and MegaBace 1000 automatic sequencers.

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: The seed-specific promoter together with the relative 5′ UTR and transit peptide sequence was amplified from soybean DNA with primers inserting an XbaI and BamHI site (forward primer: TCT AGA GTT TTC AAA TTT GAA TTT TAA TGT GTG TTG and reverse primer: GGA TCC CAC CTT AAG GAG GTT GCA ACG AGC GTG GCA). .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene. .. The engineered plasmids were introduced in Agrobacterium tumefaciens strain EHA105 by electroporation.

Article Title: Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana
Article Snippet: Paragraph title: Sequencing of BAC Clones ... After end-filling, DNA fragments were size fractionated and cloned into the Sma I site of pUC18 or Hin cII site of pUC19 (Amersham Pharmacia Biotech).

Recombinant:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: E. coli DH5α was used for the propagation of recombinant plasmid DNA. .. The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ).

Cosmid DNA:

Article Title: Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses
Article Snippet: The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega). .. The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega).

Marker:

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. The constructed mutants were designated PGAGU101 (PGN_1171::erm ), PGAGU104 (PGN_0725::erm ), PGAGU108 (PGN_1888::erm ), and PGAGU109 (PGN_1341::erm ).

Mutagenesis:

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: Paragraph title: Construction of P. gingivalis Mutant Strains ... The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan).

Isolation:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. This library was transformed into MC1061-MRL1.

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility
Article Snippet: 1 µg) received from digestion with AP1 and AP2, respectively, was extracted from agarose gels and treated with T4 DNA polymerase (0.5 units; NEB) for 10 min at 4°C in the absence of dNTPs, followed by 30 min incubation at 12°C in the presence of 0.2 mM dNTPs. .. Blunt-ended plasmids were religated either in the absence or presence of the 692 bp Dra I fragment isolated from pUC19. .. The cleavage sites of three independent clones (two linearized by AP1 and AP2, respectively, and ligated to the Dra I-fragment, and one linearized by AP2 and directly religated) were analyzed by sequencing.

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: Total RNA was extracted from 200 mg of human placenta with TRIzol Reagent (Life Technologies) and poly(A)+ fraction isolated with the polyATract mRNA Isolation System (Promega) and reverse transcribed with M-MLV enzyme (Stratagene) using specific primers for the human GAA coding sequence (GAT ATC CTA ACA CCA GCT GAC GAG AAA CTG). .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Purification:

Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility
Article Snippet: After heat inactivation (10 min, 75°C), double-stranded DNA was restricted with Bam HI/Xba I, followed by heat inactivation (20 min, 85°C) and purification on a matrix (JetSorb; GENOMED, Löhne, Germany). .. Cleaved fragments were ligated in the presence of T4 ligase (NEB) into the compatible sites of pUC19 (GE Healthcare).

Article Title: Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication
Article Snippet: The plasmid pORI(wt) has been described ( ). pUC19 was purchased from Amersham Pharmacia. .. Plasmids pORI(mut8), pORI(mut9), pORI(mut10), and pORImut(11) were constructed as described below.

Polymerase Chain Reaction:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ). .. Genomic and plasmid DNAs were prepared according to standard protocols ( ).

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The erm cassette was amplified from plasmid pVA2198 ( ). .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan). .. Ligated fragments were introduced into P. gingivalis ATCC 33277 by electroporation.

Plasmid Preparation:

Article Title: Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila, plaB, Exhibiting Hemolytic Activity
Article Snippet: E. coli DH5α was used for the propagation of recombinant plasmid DNA. .. The following vectors were used: pUC18 (backbone in plasmids pKHL102, pCL102-1, pCL102-2, pCL102-3, pKH190, pKH194, and p hlyCABD ) or pUC19 (Amersham Biosciences, Freiburg, Germany), pBCKS+ (backbone in plasmid pKH192; Stratagene, Heidelberg, Germany), and pBOC20 (backbone in plasmid pKH195) ( ). .. Genomic and plasmid DNAs were prepared according to standard protocols ( ).

Article Title: Three CoA Transferases Involved in the Production of Short Chain Fatty Acids in Porphyromonas gingivalis
Article Snippet: The erm cassette was amplified from plasmid pVA2198 ( ). .. The amplicons were joined using mixed PCR ( ) or the In-fusion HD Cloning kit (Takara Bio, Otsu, Japan), and subcloned into pUC19 (GE Healthcare Japan, Hino, Japan).

Article Title: Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses
Article Snippet: The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega). .. The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega).

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: An EcoRV site was inserted respectively in the forward and in the reverse primer to facilitate subsequent cDNA cloning in the plant expression vector. .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Article Title: Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication
Article Snippet: T65 is a 65-mer of oligodeoxythymidylate. .. The plasmid pORI(wt) has been described ( ). pUC19 was purchased from Amersham Pharmacia. .. Plasmids pORI(mut8), pORI(mut9), pORI(mut10), and pORImut(11) were constructed as described below.

Software:

Article Title: Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana
Article Snippet: After end-filling, DNA fragments were size fractionated and cloned into the Sma I site of pUC18 or Hin cII site of pUC19 (Amersham Pharmacia Biotech). .. Clones were sequenced using the ABI PRISM Dye Terminator Cycle Sequencing ready Reaction kit with FS AmpliTaq DNA polymerase (PE Applied Biosystems) and analyzed on ABI 377 (PE Applied Biosystems) sequencing gels.

RNA Extraction:

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: Paragraph title: RNA Extraction, cDNA Amplification, and Cloning ... Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

Knock-Out:

Article Title: Recognition and discrimination of target mRNAs by Sib RNAs, a cis-encoded sRNA family
Article Snippet: Strains DY330 and MG1655 were used for the construction of knockout strains. .. Plasmids pBAD/Myc -His B (Invitrogen), pACYC184, pUC19 and pKK232-8 (Amersham Pharmacia) were used as cloning vectors.

Concentration Assay:

Article Title: Short-Tailed Stx Phages Exploit the Conserved YaeT Protein To Disseminate Shiga Toxin Genes among Enterobacteria
Article Snippet: A genetic library from E. coli MC1061 was created following the ligation of partially Sau3AI-digested chromosomal DNA (2- to 5-kb fragments) into BamHI-restricted, dephosphorylated expression vectors pUC18 and pUC19 (Amersham-Pharmacia). .. Transformants were isolated on LB agar containing 100 μg ml−1 ampicillin and then used to inoculate LB-Ca.

Alkaline Lysis:

Article Title: Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses
Article Snippet: The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega). .. The genome sequence of H. seropedicae strain SmR1 total DNA was determined by the whole genome sequencing strategy using short fragment (1.5–3.0 kb) libraries in pUC18 and pUC19 (Amersham Biosciences) and cosmid libraries in Supercos (Promega).

BAC Assay:

Article Title: Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana
Article Snippet: Paragraph title: Sequencing of BAC Clones ... After end-filling, DNA fragments were size fractionated and cloned into the Sma I site of pUC18 or Hin cII site of pUC19 (Amersham Pharmacia Biotech).

CTG Assay:

Article Title: Production of a Functional Human Acid Maltase in Tobacco Seeds: Biochemical Analysis, Uptake by Human GSDII Cells, and In Vivo Studies in GAA Knockout Mice
Article Snippet: Total RNA was extracted from 200 mg of human placenta with TRIzol Reagent (Life Technologies) and poly(A)+ fraction isolated with the polyATract mRNA Isolation System (Promega) and reverse transcribed with M-MLV enzyme (Stratagene) using specific primers for the human GAA coding sequence (GAT ATC CTA ACA CCA GCT GAC GAG AAA CTG). .. Controlling elements and mature peptide sequence were assembled in pUC19 (Pharmacia-Amersham) and the whole tract cloned in pBI101 (Clontech) in place of the gusA gene.

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    GE Healthcare puc19
    Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for <t>pUC19-D).</t> Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
    Puc19, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19/product/GE Healthcare
    Average 88 stars, based on 5 article reviews
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    puc19 - by Bioz Stars, 2019-10
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    79
    GE Healthcare puc19 dna
    Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for <t>pUC19-D).</t> Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,
    Puc19 Dna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19 dna/product/GE Healthcare
    Average 79 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

    Journal: PLoS ONE

    Article Title: The Mitochondrial LSU rRNA Group II Intron of Ustilago maydis Encodes an Active Homing Endonuclease Likely Involved in Intron Mobility

    doi: 10.1371/journal.pone.0049551

    Figure Lengend Snippet: Analysis of the I- Uma I target site specificity. CF from induced pAP2 cells was incubated with various substrate plasmids under standard conditions for analysis of cleavage efficiencies (see Materials and Methods). Letters denote the individual substrate plasmids ( e.g. D for pUC19-D). Reaction products were additionally cleaved with Ssp I to yield 2.1 and 0.63 kb fragments in case of cleavage (schematic on the right: the I- Uma I target site fragment is indicated as black box. Xba I cleaves at the right border and in combination with Ssp I produces fragments similar in size to those produced by I- Uma I/ Ssp I). Marker lanes: lin. S, pUC19-D cleaved with Ssp I; lin. X/S, pUC19-D cleaved with Xba I/ Ssp I; s.c./circ., uncleaved pUC19-D showing the supercoiled and circular forms. The + and - symbols refer to the cleavage efficiencies. +++, complete cleavage; ++/+++, > 50% cleavage; ++, ∼50% cleavage; +,

    Article Snippet: Cleaved fragments were ligated in the presence of T4 ligase (NEB) into the compatible sites of pUC19 (GE Healthcare).

    Techniques: Incubation, Produced, Marker