klenow fragment  (Millipore)


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    Name:
    DNA Polymerase I
    Description:
    DNA polymerase I yields two fragments small and large upon protease digestion The large fragment Klenow fragment loses the 5 exonuclease activity that is present in the intact holoenzyme However it retains both the polymerase 5 3 activity and the 3 5 exonuclease activity of the native enzyme
    Catalog Number:
    d8276
    Price:
    None
    Applications:
    Suitable for:. DNA sequencing by the Sanger dideoxy method. Synthesis of the complementary strand of cDNA. Filling in 5'-overhangs in double stranded DNA to form blunt ends. Mutagenesis of DNA with second strand synthesis using oligonucleotides. Labeling DNA by the random primer method
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    Structured Review

    Millipore klenow fragment
    Effects of <t>lipoxin</t> A4 concentration on induction of PPAR-γ
    DNA polymerase I yields two fragments small and large upon protease digestion The large fragment Klenow fragment loses the 5 exonuclease activity that is present in the intact holoenzyme However it retains both the polymerase 5 3 activity and the 3 5 exonuclease activity of the native enzyme
    https://www.bioz.com/result/klenow fragment/product/Millipore
    Average 90 stars, based on 1419 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Mechanisms Mediating Reduced Responsiveness of Neonatal Neutrophils to Lipoxin A4"

    Article Title: Mechanisms Mediating Reduced Responsiveness of Neonatal Neutrophils to Lipoxin A4

    Journal: Pediatric research

    doi: 10.1203/PDR.0b013e318180e4af

    Effects of lipoxin A4 concentration on induction of PPAR-γ
    Figure Legend Snippet: Effects of lipoxin A4 concentration on induction of PPAR-γ

    Techniques Used: Concentration Assay

    Effects of lipoxin A4 on neutrophil respiratory burst activity
    Figure Legend Snippet: Effects of lipoxin A4 on neutrophil respiratory burst activity

    Techniques Used: Activity Assay

    Effects of lipoxin A4 on expression of PPAR-γ and NGAL
    Figure Legend Snippet: Effects of lipoxin A4 on expression of PPAR-γ and NGAL

    Techniques Used: Expressing

    Effects of lipoxin A4 on neutrophil chemotaxis and apoptosis
    Figure Legend Snippet: Effects of lipoxin A4 on neutrophil chemotaxis and apoptosis

    Techniques Used: Chemotaxis Assay

    2) Product Images from "Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum"

    Article Title: Fyn Signaling Is Compartmentalized to Dopamine D1 Receptor Expressing Neurons in the Dorsal Medial Striatum

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00273

    Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p
    Figure Legend Snippet: Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t -test. *** p

    Techniques Used: Plasmid Preparation, shRNA, Expressing, Incubation, In Vitro, Infection, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

    Related Articles

    Clone Assay:

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: .. The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment. .. The low-copy-number plasmids pIRHis and pIHisR, in which wild-type fecR was replaced by fecRhis or hisfecR , respectively, were created as follows.

    Amplification:

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: The fecR gene was amplified by PCR from plasmid pSV66 fecIRA ′ with primers FecRI and His2701 (Table ). .. The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment.

    Article Title: Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
    Article Snippet: The downstream DNA regions of the corresponding genes were PCR amplified with primer pairs waaLdown Sal I (5′-TACGCGTCGACAAACAGGCTCTCCAATCAGC-3′) and waaLdown Apa I (5′-GTAAGGGCCCGCCAAAGCAACATACCTTTCC-3′), and galKdown Sal I (5′-TACGCGTCGACCACCCGAAGCAAGTAGAAGC-3′) and galKdown Apa I (5′-GTAAGGGCCCATTTGCCAGCGACGACGATC-3′). .. We deleted the tetracycline resistance gene ( tetR ) by first digesting pSSkan wavB1 with restriction enzymes Sna BI and Nde I, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSS wavB2 , which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB.

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ). .. The recombinant pET-Int expression plasmid was amplified in a DH5α strain of E. coli (Invitrogen, USA)and sequenced with specific primer.

    Article Title: Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis
    Article Snippet: .. PCR amplification The PCR reaction was carried out in a 25-μl reaction mixture containing the following: 1 μl of DNA (50 ng/μl−1 ), 12.5 μl PCR buffer (50 mmol/l−1 KCl, 1.5 mmol/l−1 MgCl2 , 10 mmol/l−1 Tris–HCl, pH 8.8, 0.1% TritonX-100), 1U polymerase (Sigma Aldrich, St. Louis, MO, USA), 10 mmol/l−1 dNTP (Invitrogen, Carlsbad, CA, USA), 0.5 μl 100 mmol/l−1 of each primer, and 11.5 μl H2 O. Amplifications were performed in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation for 5 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 53–56 °C, 1 min at 72 °C, and a final extension for 10 min at 72 °C. .. Amplification products were separated on a 1.5% agarose gel (Invitrogen, Carlsbad, CA, USA) in 1 × TBE buffer (0.178 mol/l−1 Tris-borate, 0.178 mol/l−1 boric acid, 0.004 mmol/l−1 EDTA) and stained with ethidium bromide.

    Filtration:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. Fractions containing pol β were identified again by electrophoresis via 12.5% SDS–PAGE, pooled, and concentrated to < 5 mL before being loaded onto a gel filtration column (HiLoad 16/60 Superdex 75 prep grade, GE Healthcare Life Sciences).

    DNA Ligation:

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: .. Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ). .. The recombinant pET-Int expression plasmid was amplified in a DH5α strain of E. coli (Invitrogen, USA)and sequenced with specific primer.

    Construct:

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: Plasmid pFecRHis was constructed by inserting the fecR gene upstream of the codons encoding six histidine residues of the fusion vector pET25b(+) (Novagen, Schwalbach, Germany) cleaved with Nde I- Eco RI. .. The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment.

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: The full-length construct of rat pol β was expressed in Rosetta2 DE3 cells as previously described for similar pol β constructs. .. The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation.

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: Integrase-expressing plasmid was constructed as follows: pCMVInt vector was digested by Kpn I, and then was blunted using klenow fragment (Fermentas, Lithuania). .. Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ).

    Real-time Polymerase Chain Reaction:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    Microarray:

    Article Title: Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains
    Article Snippet: Paragraph title: Hybridisation and microarray scanning ... Two micrograms of each restriction enzyme (Taq I and Msp I) digested genomic DNA was labelled by using 20 μl of the 2.5X Random Primer, 40 U of the Klenow fragment, and 3 μl of the Cy5-dCTP or Cy3-dCTP (1 mM stocks) at 37°C for 2 h. Unincorporated fluorescent nucleotides were removed by using Microcon 30 filter columns (Millipore, Milano, Italy).

    Incubation:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: .. Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    Article Title: Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp
    Article Snippet: .. Site-specific DNA photo-crosslinking To prepare 3΄ end-labelled oligonucleotide substrates for photo-crosslinking, 45 pmol of RF-30 primer was annealed with 30 pmol of TEMP90-R to create APB-Junction or with 30 pmol of TEMP90-Rneo to create APB-End (see Table for primer sequences), and incubated with 3.1 μM [α-32 P] TTP (PerkinElmer Life Sciences), 20 μM α-S-dCTP (ChemCyte) and 4 units of Klenow fragment at 10°C for 30 min in a 20 μl Klenow reaction mixture, followed by a chase with 50 μM TTP at 10°C for 30 min. After phenol–chloroform (1:1) extraction, the product DNA was incubated with 2.1 nmol of azidophenacyl bromide (APB; Sigma) in a 50 μl volume for 3 h at room temperature in the dark. .. After removal of unreacted reagent by ethanol precipitation with ethachinmate (NIPPON GENE), the product DNA was dissolved in TE at 50 nM (calculated from the estimated recovery from incorporated TMP), and stored at 4°C in the dark.

    Expressing:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: Paragraph title: Expression and Purification of Proteins ... The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation.

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: As determined by IF analysis, the μNS antiserum did not stain cells expressing μ2 or GFP, and the GFP antibodies did not stain cells expressing μNS or μ2. .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ). .. The recombinant pET-Int expression plasmid was amplified in a DH5α strain of E. coli (Invitrogen, USA)and sequenced with specific primer.

    Modification:

    Article Title: Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis
    Article Snippet: Total DNA was isolated from embryos of different stages, and the degree of methylation in CpG-containing sequences was determined by a modified nearest-neighbor analysis ( ). .. Five to 10 μg of DNA were nicked with 10 U of DNase I (Boehringer Mannheim, Mannheim, Germany) for 15 min at 37°C and 3′-end labeled with 25 μCi of [α32 P]dGTP (3000 Ci/mmol; Amersham, Braunschweig, Germany) and 10 U DNA polymerase I (Sigma, Deisenhofen, Germany) for 30 min at 15°C in a 25-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5.0 mM CaCl2 , and 0.04% β-mercaptoethanol.

    Hybridization:

    Article Title: Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains
    Article Snippet: Paragraph title: Hybridisation and microarray scanning ... Two micrograms of each restriction enzyme (Taq I and Msp I) digested genomic DNA was labelled by using 20 μl of the 2.5X Random Primer, 40 U of the Klenow fragment, and 3 μl of the Cy5-dCTP or Cy3-dCTP (1 mM stocks) at 37°C for 2 h. Unincorporated fluorescent nucleotides were removed by using Microcon 30 filter columns (Millipore, Milano, Italy).

    Transfection:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: The μ2 antiserum showed a low level of background nuclear fluorescence with paraformaldehyde (PFA) fixation that was reduced with methanol fixation as previously described , but the antiserum did not stain μNS or GFP in transfected cells. .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Ligation:

    Article Title: Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
    Article Snippet: The ligation mix was digested with Sma I and Apa I, purified, and ligated into pKEK229 ( ) that had been digested with Sma I and Apa I to give plasmids pKEKΔ galK and pKEKΔ waaL . .. We deleted the tetracycline resistance gene ( tetR ) by first digesting pSSkan wavB1 with restriction enzymes Sna BI and Nde I, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSS wavB2 , which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB.

    Article Title: Ionic effects on the elasticity of single DNA molecules
    Article Snippet: The 5′-overhangs of λ DNA (methylated c 1857 ind 1 Sam 7; New England Biolabs) were biotinylated with the Klenow fragment of DNA polymerase using biotin-11-dCTP (Sigma), dATP, dGTP, and dUTP as described previously ( ). .. After biotinylation and nick ligation, DNA stocks were stored in an EDTA-containing buffer.

    Protease Inhibitor:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: .. The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. The protein mixture was filtered with 5 and 0.45 μ m syringe filters before being loaded onto a HiTrap Heparin column and separated by a NaCl gradient [buffer A, 25 mM HEPES, 100 mM NaCl, and 5% glycerol (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)] using a liquid chromatography system (Aekta Prime, Amersham Biosciences).

    Low Copy Number:

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment. .. The low-copy-number plasmids pIRHis and pIHisR, in which wild-type fecR was replaced by fecRhis or hisfecR , respectively, were created as follows.

    Liquid Chromatography:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. The protein mixture was filtered with 5 and 0.45 μ m syringe filters before being loaded onto a HiTrap Heparin column and separated by a NaCl gradient [buffer A, 25 mM HEPES, 100 mM NaCl, and 5% glycerol (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)] using a liquid chromatography system (Aekta Prime, Amersham Biosciences).

    Generated:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen). .. In brief, expression was induced with 1 mM isopropyl-β- d -thiogalactopyranoside.

    other:

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine
    Article Snippet: Escherichia coli exonuclease III was obtained from Epicentre Technologies (Madison, WI), and the Klenow fragment of E.coli DNA polymerase I was purchased from Sigma (St Louis, MO).

    DNA Labeling:

    Article Title: Large-scale genetic variation of the symbiosis-required megaplasmid pSymA revealed by comparative genomic analysis of Sinorhizobium meliloti natural strains
    Article Snippet: Genomic DNA was labelled with FluoroLink Cy3- or Cy5-dCTP (Amersham Biosciences, Milano, Italy) by using the method described by Pollack et al. [ ] and the components of the BioPrime DNA labeling system (Invitrogen, Milano, Italy). .. Two micrograms of each restriction enzyme (Taq I and Msp I) digested genomic DNA was labelled by using 20 μl of the 2.5X Random Primer, 40 U of the Klenow fragment, and 3 μl of the Cy5-dCTP or Cy3-dCTP (1 mM stocks) at 37°C for 2 h. Unincorporated fluorescent nucleotides were removed by using Microcon 30 filter columns (Millipore, Milano, Italy).

    Polymerase Chain Reaction:

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: The fecR gene was amplified by PCR from plasmid pSV66 fecIRA ′ with primers FecRI and His2701 (Table ). .. The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment.

    Article Title: Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
    Article Snippet: The upstream and downstream PCR products of waaL and galK were digested with Sal I and ligated overnight. .. We deleted the tetracycline resistance gene ( tetR ) by first digesting pSSkan wavB1 with restriction enzymes Sna BI and Nde I, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSS wavB2 , which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB.

    Article Title: Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis
    Article Snippet: .. PCR amplification The PCR reaction was carried out in a 25-μl reaction mixture containing the following: 1 μl of DNA (50 ng/μl−1 ), 12.5 μl PCR buffer (50 mmol/l−1 KCl, 1.5 mmol/l−1 MgCl2 , 10 mmol/l−1 Tris–HCl, pH 8.8, 0.1% TritonX-100), 1U polymerase (Sigma Aldrich, St. Louis, MO, USA), 10 mmol/l−1 dNTP (Invitrogen, Carlsbad, CA, USA), 0.5 μl 100 mmol/l−1 of each primer, and 11.5 μl H2 O. Amplifications were performed in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation for 5 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 53–56 °C, 1 min at 72 °C, and a final extension for 10 min at 72 °C. .. Amplification products were separated on a 1.5% agarose gel (Invitrogen, Carlsbad, CA, USA) in 1 × TBE buffer (0.178 mol/l−1 Tris-borate, 0.178 mol/l−1 boric acid, 0.004 mmol/l−1 EDTA) and stained with ethidium bromide.

    Sonication:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: .. The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. The protein mixture was filtered with 5 and 0.45 μ m syringe filters before being loaded onto a HiTrap Heparin column and separated by a NaCl gradient [buffer A, 25 mM HEPES, 100 mM NaCl, and 5% glycerol (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)] using a liquid chromatography system (Aekta Prime, Amersham Biosciences).

    Binding Assay:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    ChIP-sequencing:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    Nucleic Acid Electrophoresis:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen). .. To further purify μ2 from the insoluble fraction, it was subjected to preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroeluted ( ).

    Fluorescence:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: The μ2 antiserum showed a low level of background nuclear fluorescence with paraformaldehyde (PFA) fixation that was reduced with methanol fixation as previously described , but the antiserum did not stain μNS or GFP in transfected cells. .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Methylation:

    Article Title: Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis
    Article Snippet: Total DNA was isolated from embryos of different stages, and the degree of methylation in CpG-containing sequences was determined by a modified nearest-neighbor analysis ( ). .. Five to 10 μg of DNA were nicked with 10 U of DNase I (Boehringer Mannheim, Mannheim, Germany) for 15 min at 37°C and 3′-end labeled with 25 μCi of [α32 P]dGTP (3000 Ci/mmol; Amersham, Braunschweig, Germany) and 10 U DNA polymerase I (Sigma, Deisenhofen, Germany) for 30 min at 15°C in a 25-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5.0 mM CaCl2 , and 0.04% β-mercaptoethanol.

    Article Title: Ionic effects on the elasticity of single DNA molecules
    Article Snippet: .. The 5′-overhangs of λ DNA (methylated c 1857 ind 1 Sam 7; New England Biolabs) were biotinylated with the Klenow fragment of DNA polymerase using biotin-11-dCTP (Sigma), dATP, dGTP, and dUTP as described previously ( ). ..

    Isolation:

    Article Title: Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis
    Article Snippet: Total DNA was isolated from embryos of different stages, and the degree of methylation in CpG-containing sequences was determined by a modified nearest-neighbor analysis ( ). .. Five to 10 μg of DNA were nicked with 10 U of DNase I (Boehringer Mannheim, Mannheim, Germany) for 15 min at 37°C and 3′-end labeled with 25 μCi of [α32 P]dGTP (3000 Ci/mmol; Amersham, Braunschweig, Germany) and 10 U DNA polymerase I (Sigma, Deisenhofen, Germany) for 30 min at 15°C in a 25-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5.0 mM CaCl2 , and 0.04% β-mercaptoethanol.

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: .. Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    Labeling:

    Article Title: Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis
    Article Snippet: .. Five to 10 μg of DNA were nicked with 10 U of DNase I (Boehringer Mannheim, Mannheim, Germany) for 15 min at 37°C and 3′-end labeled with 25 μCi of [α32 P]dGTP (3000 Ci/mmol; Amersham, Braunschweig, Germany) and 10 U DNA polymerase I (Sigma, Deisenhofen, Germany) for 30 min at 15°C in a 25-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5.0 mM CaCl2 , and 0.04% β-mercaptoethanol. .. After termination by 10 mM EDTA, nonincorporated dGTP was removed using a Sephadex G-50 minicolumn (Boehringer Mannheim).

    Mouse Assay:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Approximately 200 mg of frozen liver tissue were used for chromatin isolation from three mice of each group. .. Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation.

    Article Title: Respiratory hyperoxia reverses immunosuppression by regulating myeloid-derived suppressor cells and PD-L1 expression in a triple-negative breast cancer mouse model
    Article Snippet: In brief, on day 7, 14, or 21, 4T1 tumor-bearing mice were sacrificed, and their lungs were harvested. .. Lung samples were finely minced and digested in 5 ml of an enzyme cocktail containing 1 × RPMI-1640, 2% (v/v) FBS, 0.002% (w/v) collagenase D (Sigma-Aldrich; Saint Louis, MO, USA) and 0.002% (w/v) DNA polymerase I (Sigma-Aldrich) for 1 hour at 37°C.

    Sequencing:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Primers were designed for the site that contains the highest binding frequency, the largest coverage area, and the HNF4α binding sequence.

    Positron Emission Tomography:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen). .. In brief, expression was induced with 1 mM isopropyl-β- d -thiogalactopyranoside.

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ). .. The recombinant pET-Int expression plasmid was amplified in a DH5α strain of E. coli (Invitrogen, USA)and sequenced with specific primer.

    Immunoprecipitation:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: .. Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation. .. Real-time PCR analysis was done using primers for the putative HNF4α binding sites identified by ChIP-Seq data analysis described above for Ect2, Osgin1 , and Hjurp ( ).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen). .. To further purify μ2 from the insoluble fraction, it was subjected to preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electroeluted ( ).

    Purification:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: .. The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. The protein mixture was filtered with 5 and 0.45 μ m syringe filters before being loaded onto a HiTrap Heparin column and separated by a NaCl gradient [buffer A, 25 mM HEPES, 100 mM NaCl, and 5% glycerol (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)] using a liquid chromatography system (Aekta Prime, Amersham Biosciences).

    Article Title: Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
    Article Snippet: The ligation mix was digested with Sma I and Apa I, purified, and ligated into pKEK229 ( ) that had been digested with Sma I and Apa I to give plasmids pKEKΔ galK and pKEKΔ waaL . .. We deleted the tetracycline resistance gene ( tetR ) by first digesting pSSkan wavB1 with restriction enzymes Sna BI and Nde I, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSS wavB2 , which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB.

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: We used rabbit polyclonal antisera against μNS ( ) and μ2 (described below). μNS and μ2 polyclonal IgG antibodies purified with protein A-Sepharose were directly conjugated to Texas red and Oregon green by following the manufacturer's procedure (Molecular Probes). .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Chromatin Immunoprecipitation:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: Paragraph title: ChIP. ... Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation.

    SDS Page:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. Fractions containing pol β were identified by electrophoresis on a 12.5% SDS–PAGE gel, pooled, diluted 1:5 with 25 mM HEPES and 25 mM NaCl (pH 7.0), loaded onto an SP Sepharose column, and again separated by a NaCl gradient [buffer C, 25 mM HEPES and 25 mM NaCl (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)].

    Plasmid Preparation:

    Article Title: Hepatocyte-specific deletion of hepatocyte nuclear factor-4? in adult mice results in increased hepatocyte proliferation
    Article Snippet: ChIP was done using whole liver tissue from HNF4αfl/fl mice treated with the MUP-iCre-AAV8 vector and from HNF4αfl/fl mice treated with the MUP-EGFP-AAV8 control vector and a protocol adapted from a method previously published by Buchholz et al. ( ). .. Isolated chromatin was then incubated with mouse IgG (1 μg; Millipore), DNA polymerase II (1 μg; Millipore), and HNF4α (10 μg; R & D Systems) antibodies for immunoprecipitation.

    Article Title: Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins
    Article Snippet: .. The Nde I- Hin dIII fragment of fecR from plasmid pAA70 was blunt ended with the Klenow fragment of DNA polymerase I, cloned downstream of the codons encoding 10 histidine residues in the fusion vector pET19b (Novagen), cleaved with Nde I- Bam HI, and blunt ended with the Klenow fragment. .. The low-copy-number plasmids pIRHis and pIHisR, in which wild-type fecR was replaced by fecRhis or hisfecR , respectively, were created as follows.

    Article Title: Role of Vibrio cholerae O139 Surface Polysaccharides in Intestinal Colonization
    Article Snippet: .. We deleted the tetracycline resistance gene ( tetR ) by first digesting pSSkan wavB1 with restriction enzymes Sna BI and Nde I, then treating with Klenow fragment (Sigma), and religating, yielding plasmid pSS wavB2 , which had lost most of the tetR gene (341-bp internal fragment) and constitutively expressed wavB. .. The chromosomal mutations in V. cholerae O139 strain MO10 were constructed with different suicide plasmids (Table ) as described previously ( , ).

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: At the same time, pET15b plasmid was digested by Nde I. .. Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ).

    Electrophoresis:

    Article Title: Structural Changes in the Hydrophobic Hinge Region Adversely Affect the Activity and Fidelity of the I260Q Mutator DNA Polymerase β
    Article Snippet: The purification of wild type and I260Q pol β is based on a published purification protocol of Klenow fragment., Briefly, the cells were resuspended in 25 mM HEPES (pH 7.0), 100 mM NaCl, and 5% glycerol with lysozyme in the presence of Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich), and sonicated (5 s pulse × 30 s rest) for 10 min while they were suspended in an ice/water bath, followed by ultracentrifugation. .. Fractions containing pol β were identified by electrophoresis on a 12.5% SDS–PAGE gel, pooled, diluted 1:5 with 25 mM HEPES and 25 mM NaCl (pH 7.0), loaded onto an SP Sepharose column, and again separated by a NaCl gradient [buffer C, 25 mM HEPES and 25 mM NaCl (pH 7.0); buffer B, 25 mM HEPES and 2 M NaCl (pH 7.0)].

    Recombinant:

    Article Title: Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli
    Article Snippet: Then, it was blunted by klenow fragment, and digested with Bam HI. pET15b (Novagen, CA, USA), backbone and integrase open reading frame, were extracted from the gel and ligated using DNA Ligation Kit (TaKaRa, Japan) ( ). .. The recombinant pET-Int expression plasmid was amplified in a DH5α strain of E. coli (Invitrogen, USA)and sequenced with specific primer.

    Agarose Gel Electrophoresis:

    Article Title: Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis
    Article Snippet: PCR amplification The PCR reaction was carried out in a 25-μl reaction mixture containing the following: 1 μl of DNA (50 ng/μl−1 ), 12.5 μl PCR buffer (50 mmol/l−1 KCl, 1.5 mmol/l−1 MgCl2 , 10 mmol/l−1 Tris–HCl, pH 8.8, 0.1% TritonX-100), 1U polymerase (Sigma Aldrich, St. Louis, MO, USA), 10 mmol/l−1 dNTP (Invitrogen, Carlsbad, CA, USA), 0.5 μl 100 mmol/l−1 of each primer, and 11.5 μl H2 O. Amplifications were performed in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation for 5 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 53–56 °C, 1 min at 72 °C, and a final extension for 10 min at 72 °C. .. Amplification products were separated on a 1.5% agarose gel (Invitrogen, Carlsbad, CA, USA) in 1 × TBE buffer (0.178 mol/l−1 Tris-borate, 0.178 mol/l−1 boric acid, 0.004 mmol/l−1 EDTA) and stained with ethidium bromide.

    Ethanol Precipitation:

    Article Title: Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp
    Article Snippet: Site-specific DNA photo-crosslinking To prepare 3΄ end-labelled oligonucleotide substrates for photo-crosslinking, 45 pmol of RF-30 primer was annealed with 30 pmol of TEMP90-R to create APB-Junction or with 30 pmol of TEMP90-Rneo to create APB-End (see Table for primer sequences), and incubated with 3.1 μM [α-32 P] TTP (PerkinElmer Life Sciences), 20 μM α-S-dCTP (ChemCyte) and 4 units of Klenow fragment at 10°C for 30 min in a 20 μl Klenow reaction mixture, followed by a chase with 50 μM TTP at 10°C for 30 min. After phenol–chloroform (1:1) extraction, the product DNA was incubated with 2.1 nmol of azidophenacyl bromide (APB; Sigma) in a 50 μl volume for 3 h at room temperature in the dark. .. After removal of unreacted reagent by ethanol precipitation with ethachinmate (NIPPON GENE), the product DNA was dissolved in TE at 50 nM (calculated from the estimated recovery from incorporated TMP), and stored at 4°C in the dark.

    Produced:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: Rabbit polyclonal antiserum specific for μ2 was produced by using Escherichia coli -expressed protein. .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Concentration Assay:

    Article Title: Ionic effects on the elasticity of single DNA molecules
    Article Snippet: The 5′-overhangs of λ DNA (methylated c 1857 ind 1 Sam 7; New England Biolabs) were biotinylated with the Klenow fragment of DNA polymerase using biotin-11-dCTP (Sigma), dATP, dGTP, and dUTP as described previously ( ). .. Monovalent salt solutions were prepared by diluting 100 mM cacodylate pH 7 buffer stocks (86.2 mM sodium cacodylate, 13.8 mM cacodylic acid) supplemented with either 100 or 500 mM NaCl (total Na+ concentration ca.

    Thin Layer Chromatography:

    Article Title: Nuclear Accumulation of S-Adenosylhomocysteine Hydrolase in Transcriptionally Active Cells during Development of Xenopus laevis
    Article Snippet: Five to 10 μg of DNA were nicked with 10 U of DNase I (Boehringer Mannheim, Mannheim, Germany) for 15 min at 37°C and 3′-end labeled with 25 μCi of [α32 P]dGTP (3000 Ci/mmol; Amersham, Braunschweig, Germany) and 10 U DNA polymerase I (Sigma, Deisenhofen, Germany) for 30 min at 15°C in a 25-μl reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5.0 mM CaCl2 , and 0.04% β-mercaptoethanol. .. The resulting desoxynucleoside 3′-monophosphates were applied to cellulose TLC sheets (Machery-Nagel, Düren, Germany) and separated in two dimensions ( ).

    Staining:

    Article Title: Mammalian Reovirus Nonstructural Protein ?NS Forms Large Inclusions and Colocalizes with Reovirus Microtubule-Associated Protein ?2 in Transfected Cells
    Article Snippet: The μ2 antiserum showed a low level of background nuclear fluorescence with paraformaldehyde (PFA) fixation that was reduced with methanol fixation as previously described , but the antiserum did not stain μNS or GFP in transfected cells. .. The T1L M1 gene was excised from pBluescript II KS(+) (Stratagene, La Jolla, Calif.) ( ) with Sma I and Xho I and ligated to pET-21b (Novagen, Madison, Wis.) cut with Hin dIII and Xho I, with the Hin dIII overhang converted to a blunt end with the Klenow fragment of DNA polymerase I; this procedure generated pET-M1(T1L). μ2 was expressed in BL21-DE3 cells (Novagen) by following the procedure in the pET system manual (Novagen).

    Article Title: Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis
    Article Snippet: PCR amplification The PCR reaction was carried out in a 25-μl reaction mixture containing the following: 1 μl of DNA (50 ng/μl−1 ), 12.5 μl PCR buffer (50 mmol/l−1 KCl, 1.5 mmol/l−1 MgCl2 , 10 mmol/l−1 Tris–HCl, pH 8.8, 0.1% TritonX-100), 1U polymerase (Sigma Aldrich, St. Louis, MO, USA), 10 mmol/l−1 dNTP (Invitrogen, Carlsbad, CA, USA), 0.5 μl 100 mmol/l−1 of each primer, and 11.5 μl H2 O. Amplifications were performed in a C1000 Touch™ Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the following conditions: initial denaturation for 5 min at 94 °C, 35 cycles of 45 s at 94 °C, 45 s at 53–56 °C, 1 min at 72 °C, and a final extension for 10 min at 72 °C. .. Amplification products were separated on a 1.5% agarose gel (Invitrogen, Carlsbad, CA, USA) in 1 × TBE buffer (0.178 mol/l−1 Tris-borate, 0.178 mol/l−1 boric acid, 0.004 mmol/l−1 EDTA) and stained with ethidium bromide.

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    Millipore klenow fragment
    Klenow Fragment, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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