t4 dna polymerase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 dna polymerase
    Single-stranded DNA ligation with <t>T4</t> DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.
    T4 Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/Thermo Fisher
    Average 96 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-01
    96/100 stars

    Images

    1) Product Images from "Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase"

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx033

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.
    Figure Legend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Techniques Used: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.
    Figure Legend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Techniques Used: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.
    Figure Legend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Techniques Used: Ligation, Sequencing, Ancient DNA Assay

    2) Product Images from "Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development"

    Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development

    Journal: The Journal of Experimental Medicine

    doi:

    ( A ) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area ). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer ( BW ) and a set of locus-specific primers (1, 2 or 1′, 2′). ( B ) SBE in developing B cells from wild-type and Eμ- myc mice. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c and Eμ- myc mice was subjected to LMPCR to detect SBE upstream of the D FL16.1 , J κ1 , and J κ2 segments. PCR products were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The identities of the indicated PCR products were confirmed by DNA sequence analysis ( 28 ). Labeled products were visualized with a PhosphorImager. Lanes 1–10 , B cell development stages as defined in Table 1 ; lane 11 , 6312, a pro-B cell line derived from a RAG-2-deficient mouse ( 3 ); lane 12 , B220 + cells from bone marrow, representing all stages of B cell development; lane 13 , same as lane 12 , with no ligase added at the linker ligation step. The bottom strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus ( CD14 ), showing equivalent amounts of amplifiable DNA in all samples.
    Figure Legend Snippet: ( A ) Diagram of the LMPCR assay used to detect signal end and coding end double stranded DNA breaks at rearranging loci. A rearranging locus is shown, with the RSS abutting the coding portion of a rearranging gene segment (J1, filled area ). Cleavage by V(D)J recombinase at an RSS generates two kinds of ends: a signal end and a coding end. The signal end is blunt and 5′-phosphorylated and available for linker ligation. The coding end is processed through a hairpin intermediate and the asterisk next to it signifies heterogeneity in the fine structure of the opened hairpin. Coding ends are blunted with T4 DNA polymerase and then subjected to linker ligation. Amplification is subsequently carried out using a linker-specific primer ( BW ) and a set of locus-specific primers (1, 2 or 1′, 2′). ( B ) SBE in developing B cells from wild-type and Eμ- myc mice. Purified DNA from sorted subpopulations of B cells from wild-type Balb/c and Eμ- myc mice was subjected to LMPCR to detect SBE upstream of the D FL16.1 , J κ1 , and J κ2 segments. PCR products were analyzed by electrophoresis on agarose gels, blot transfer to a nylon membrane, and hybridization with locus-specific oligonucleotide probes. The identities of the indicated PCR products were confirmed by DNA sequence analysis ( 28 ). Labeled products were visualized with a PhosphorImager. Lanes 1–10 , B cell development stages as defined in Table 1 ; lane 11 , 6312, a pro-B cell line derived from a RAG-2-deficient mouse ( 3 ); lane 12 , B220 + cells from bone marrow, representing all stages of B cell development; lane 13 , same as lane 12 , with no ligase added at the linker ligation step. The bottom strip shows an ethidium bromide-stained agarose gel of a control amplification of a non-rearranging locus ( CD14 ), showing equivalent amounts of amplifiable DNA in all samples.

    Techniques Used: Ligation, Amplification, Mouse Assay, Purification, Polymerase Chain Reaction, Electrophoresis, Hybridization, Sequencing, Labeling, Derivative Assay, Stripping Membranes, Staining, Agarose Gel Electrophoresis

    Related Articles

    Clone Assay:

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    Article Snippet: A 2.2-kb PstI-PvuII fragment from pAtcars4 (Table ) containing part of the chromosomally encoded arsenic resistance operon was cloned into the PstI and blunted SacI sites of pOT to give pOTars (Fig. ). .. This construct was digested with KpnI, which removed two ±200-bp fragments from the center of the arsB gene, which were later used as a probe for detecting single- and double-crossover mutants, and the ends were filled in using the T4 DNA polymerase (Fermentas, Vilnius, Lithuania).

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    Article Snippet: .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen). ..

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    Article Snippet: Paragraph title: Sequence Analysis and Cloning. ... To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Article Title: Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736
    Article Snippet: Endonucleases, T4 DNA polymerase, and T4 DNA ligase were purchased from Invitrogen (Carlsbad, Calif.) and New England Biolabs (Beverly, Mass.). .. DNA fragments for cloning were isolated after gel purification with the Agarace enzyme (Promega, Madison, Wis.) according to the manufacturer's recommendations.

    Centrifugation:

    Article Title: Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation ▿
    Article Snippet: Cells of strain CNB-1, grown at 30°C in 100 ml LB broth, were collected by centrifugation at 10,000 × g for 5 min and were resuspended in 0.8 ml of chilled solution I (50 mM glucose, 10 mM EDTA [pH 8.0], 25 mM Tris-HCl [pH 8.0]). .. The fragments were end blunted by T4 DNA polymerase (Fermentas) and ligated into linear vector pUC18.

    Amplification:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: Paragraph title: Sample procurement and RNA amplification: ... The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water.

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1). .. The Amp resistance gene fragment was amplified by PCR from the plasmid puc19 with the phosphorylated primers (5-AAACGCGCGAGACGAAAGGG-3′ and 5-GGGGTCTGACGCTCAGTGGA-3′).

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    Article Snippet: For all 55 strains, we PCR amplified and sequenced six chromosomal loci. .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen).

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
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    DNA Ligation:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen). .. Plasmid DNA and promoter DNA fragment ligation using T4 DNA ligation (Invitrogen).

    Synthesized:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: First-strand cDNA was synthesized by addition of random hexamer primers and avian myeloblastosis virus reverse transcriptase. .. Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis.

    Construct:

    Article Title: Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange ▿
    Article Snippet: .. This construct was digested with KpnI, which removed two ±200-bp fragments from the center of the arsB gene, which were later used as a probe for detecting single- and double-crossover mutants, and the ends were filled in using the T4 DNA polymerase (Fermentas, Vilnius, Lithuania). .. The ±1.45-kb XbaI-XhoI fragment from pSKM2 carrying the kanamycin resistance cassette from Tn 5 was also blunt-end cloned into the blunted KpnI sites of arsB to give p arsB ::Km (Fig. ).

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: To construct the rAQP5 tissue-specific promoter into the plasmid expression vector, the following common recombinant DNA techniques were used. .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated. .. Egf1.0R51A and Egf1.0R51K were generated from the modified bacterial expression constructs by overlap extension PCR using the gene-specific primers 5′-GTAATAACAAACTATGCTAC GCG TTCCAGCAATTCTG-3′ (forward) and 5′GCTGGAA CGC GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51A ), 5′-GTAATAACAAACTATGCTAC AAG TTCCAGCAATTCTG-3′ (forward) and 5′-GCTGGAA CTT GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51K ) (mutated Arg underlined), and vector-specific primers 5′-AGCGGATAACAATTCCCCTCTAGAAATAAT-3′ (forward) and 5′-GCTTTGTTAGCAGCCGGATCTCAGTGGT-3′ (reverse).

    Adsorption:

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    Article Snippet: .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene). .. Purified plasmid DNA was sequenced by using the dideoxy termination method ( ) with Sequenase (U.S. Biochemical).

    Microarray:

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
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    Random Hexamer Labeling:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
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    Expressing:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
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    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis. .. The adapter-ligated cDNA was recovered and ligated into BstxI-digested expression plasmid pcDNAI (Invitrogen).

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: To construct the rAQP5 tissue-specific promoter into the plasmid expression vector, the following common recombinant DNA techniques were used. .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
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    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: For bacterial expression, cDNAs were PCR-amplified using primers lacking SP sequences and directly cloned in frame with the C-terminal His tag into pET-32 Ek/LIC (Novagen). .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Modification:

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated. .. Egf1.0R51A and Egf1.0R51K were generated from the modified bacterial expression constructs by overlap extension PCR using the gene-specific primers 5′-GTAATAACAAACTATGCTAC GCG TTCCAGCAATTCTG-3′ (forward) and 5′GCTGGAA CGC GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51A ), 5′-GTAATAACAAACTATGCTAC AAG TTCCAGCAATTCTG-3′ (forward) and 5′-GCTGGAA CTT GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51K ) (mutated Arg underlined), and vector-specific primers 5′-AGCGGATAACAATTCCCCTCTAGAAATAAT-3′ (forward) and 5′-GCTTTGTTAGCAGCCGGATCTCAGTGGT-3′ (reverse).

    Transformation Assay:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen). .. Transformation using MAX Efficiency DH5α Chemically Competent Cells (Invitrogen), then culture in a shaker at 37°C for 1 hour, streak out competent cells on an LB agar or agarose ampcillin plate and grow the bacterial overnight at 37°C to obtain colonies Colony screening.

    Derivative Assay:

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: Five random genomic fragments were derived by shotgun sequencing of total genomic DNA from strain Xav19 ( ). .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen).

    Hybridization:

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
    Article Snippet: Technical procedures for microarray analysis, including quality control of RNA, labeling, hybridization and scanning of the arrays were performed by the Hartwell Center for Bioinformatics & Biotechnology (HC) at St. Jude Children's Research Hospital (SJCRH) according to standard operating procedures for Affymetrix protocols (GeneChip® Expression Analysis manual, Affymetrix, Santa Clara CA, USA). .. Samples were then used to prepare the 1st strand cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix) containing SuperScript II followed by the 2nd strand cDNA synthesis with T4 DNA polymerase. cDNA was cleaned using cDNA Cleanup Spin Column (Affymetrix), and biotin-labeled cRNA was prepared using the Gene Chip IVT Labeling Kit (Affymetrix).

    Lab-on-a-Chip:

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
    Article Snippet: Prior to their use, RNA sample integrity was analyzed with the 2100 Bioanalyzer Lab-on-a-chip system (Agilent Technologies, Santa Clara, CA, USA). .. Samples were then used to prepare the 1st strand cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix) containing SuperScript II followed by the 2nd strand cDNA synthesis with T4 DNA polymerase. cDNA was cleaned using cDNA Cleanup Spin Column (Affymetrix), and biotin-labeled cRNA was prepared using the Gene Chip IVT Labeling Kit (Affymetrix).

    Ligation:

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase
    Article Snippet: Second, an additional 45°C wash step was introduced to remove the splinter oligonucleotides after immobilization of the ligation products on streptavidin-coated beads. .. For fill-in with T4 DNA polymerase a 50 μl reaction mix was prepared containing 1× T4 DNA polymerase buffer (ThermoFisher Scientific), 0.05% Tween-20, 100 μM each dNTP, 100 pmol primer CL130 and 2 μl 5 U/μl T4 DNA polymerase (ThermoFisher Scientific).

    Article Title: Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens
    Article Snippet: .. DNA extracts were blunt-ended for adapter ligation using a 1 × 20 μL master mix of 9.5 μL of H2 O, 8 μL of 5× reaction buffer for T4 DNA polymerase, 2.5 U of T4 DNA polymerase (Fermentas) in the presence of 0.4 mM dNTPs. .. Twenty microliters of the master mix was added to 20 μL of museum DNA extract and incubated for 10 min at 70°C.

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen). .. Plasmid DNA and promoter DNA fragment ligation using T4 DNA ligation (Invitrogen).

    Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development
    Article Snippet: DNA purification, T4 polymerase polishing, and linker ligation were all carried out in agarose plugs, as described elsewhere (Schlissel, M.S., manuscript in preparation). .. After the plugs solidified, they were incubated overnight at 55°C (in 100 mM Tris, pH 8.0, 25 mM EDTA, 1% sarkosyl, and 400 μg/ml proteinase K), washed in TE containing 0.5 mM PMSF for 30 min, then washed three more times in TE over 24 h. The plugs were next treated with T4 DNA polymerase by adding in a 40 μl reaction mixture containing polymerase buffer, three units T4 DNA polymerase (Life Technologies), and 100 μM dNTPs, for 1 h at 37°C.

    Purification:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water. .. Samples were purified using RNeasy mini kit (QIAGEN), eluted in 50 μl H2 O, quantitated by OD260 , and 400 ng cRNA in 12 μl H2 O was subjected to a second round of amplification.

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: The supernatant was concentrated and purified by Ultra-0.5 centrifugal filter devices (Amicon). .. A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1).

    Article Title: Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens
    Article Snippet: DNA extracts were blunt-ended for adapter ligation using a 1 × 20 μL master mix of 9.5 μL of H2 O, 8 μL of 5× reaction buffer for T4 DNA polymerase, 2.5 U of T4 DNA polymerase (Fermentas) in the presence of 0.4 mM dNTPs. .. Following incubation, extracts were purified with CentriSpin 20 columns (Princeton Separations) returning ∼32 μL of product from a 40-μL elution, stored on ice, and immediately followed by ligation of adapters.

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen). ..

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
    Article Snippet: Samples were then used to prepare the 1st strand cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix) containing SuperScript II followed by the 2nd strand cDNA synthesis with T4 DNA polymerase. cDNA was cleaned using cDNA Cleanup Spin Column (Affymetrix), and biotin-labeled cRNA was prepared using the Gene Chip IVT Labeling Kit (Affymetrix). .. Labeled cRNA was purified with Cleanup Spin Column (Affymetrix), quantified, fragmented and spiked with biotin-labeled cRNA for bioB (1,5 pM), bioC (5 pM), bioD (25 pM) and Crex (100 pM) (GeneChip® Eukaryotic Hybridization Control Kit, Affymetrix).

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene). .. Purified plasmid DNA was sequenced by using the dideoxy termination method ( ) with Sequenase (U.S. Biochemical).

    Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development
    Article Snippet: Paragraph title: Purification of DNA for LMPCR. ... After the plugs solidified, they were incubated overnight at 55°C (in 100 mM Tris, pH 8.0, 25 mM EDTA, 1% sarkosyl, and 400 μg/ml proteinase K), washed in TE containing 0.5 mM PMSF for 30 min, then washed three more times in TE over 24 h. The plugs were next treated with T4 DNA polymerase by adding in a 40 μl reaction mixture containing polymerase buffer, three units T4 DNA polymerase (Life Technologies), and 100 μM dNTPs, for 1 h at 37°C.

    Generated:

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: Egf1.0ΔRD was generated by using the same forward primer for Egf1.0 and the reverse primer (360) 5′- GAGGAGAAGCCCGG TGCATTGTTGGCACTGGATG-3′ (344), whereas Egf1.0ΔCD was generated by using (403) 5′- GACGACGACAAG ATGGCACATGATTCGGTTTCACATAC-3′ (422) and (760) 5′- GAGGAGAAGCCCGG TCGTCGATTTAAACTTCTGCTG-3′ (740). .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Protein Concentration:

    Article Title: Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity
    Article Snippet: Protein concentration was determined by the method of Lowry [ ] with BSA as a standard. .. PCR reagents and T4 DNA polymerase were obtained from Invitrogen (San Diego, CA, U.S.A.).

    Sequencing:

    Article Title: Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation ▿
    Article Snippet: Paragraph title: Plasmid DNA isolation, sequencing, and assembly. ... The fragments were end blunted by T4 DNA polymerase (Fermentas) and ligated into linear vector pUC18.

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: Paragraph title: Fosmid paired-end sequencing library construction ... A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1).

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: Paragraph title: Sequence Analysis and Cloning. ... To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Recombinant:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: To construct the rAQP5 tissue-specific promoter into the plasmid expression vector, the following common recombinant DNA techniques were used. .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    DNA Extraction:

    Article Title: Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation ▿
    Article Snippet: Paragraph title: Plasmid DNA isolation, sequencing, and assembly. ... The fragments were end blunted by T4 DNA polymerase (Fermentas) and ligated into linear vector pUC18.

    Article Title: Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736
    Article Snippet: Paragraph title: DNA isolation and manipulation. ... Endonucleases, T4 DNA polymerase, and T4 DNA ligase were purchased from Invitrogen (Carlsbad, Calif.) and New England Biolabs (Beverly, Mass.).

    Isolation:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: mRNA was isolated from approximately 1 × 108 Vero cells. .. Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis.

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: One microgram of total cellular RNA isolated from untreated rat small intestines was reverse transcribed by using the 3′ RACE (rapid amplification of cDNA ends) System (GIBCO/BRL) according to the manufacturer’s instructions. .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene).

    Article Title: Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736
    Article Snippet: Endonucleases, T4 DNA polymerase, and T4 DNA ligase were purchased from Invitrogen (Carlsbad, Calif.) and New England Biolabs (Beverly, Mass.). .. DNA fragments for cloning were isolated after gel purification with the Agarace enzyme (Promega, Madison, Wis.) according to the manufacturer's recommendations.

    Subcloning:

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated. .. After subcloning and digestion with XbaI and HindIII, sequences were cloned into pET-32 Ek/LIC.

    Labeling:

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
    Article Snippet: .. Samples were then used to prepare the 1st strand cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix) containing SuperScript II followed by the 2nd strand cDNA synthesis with T4 DNA polymerase. cDNA was cleaned using cDNA Cleanup Spin Column (Affymetrix), and biotin-labeled cRNA was prepared using the Gene Chip IVT Labeling Kit (Affymetrix). .. Labeled cRNA was purified with Cleanup Spin Column (Affymetrix), quantified, fragmented and spiked with biotin-labeled cRNA for bioB (1,5 pM), bioC (5 pM), bioD (25 pM) and Crex (100 pM) (GeneChip® Eukaryotic Hybridization Control Kit, Affymetrix).

    Mouse Assay:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: KitlSl /KitlSl-d male mice were purchased from the Jackson Laboratories. .. The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water.

    Polymerase Chain Reaction:

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1). .. The Amp resistance gene fragment was amplified by PCR from the plasmid puc19 with the phosphorylated primers (5-AAACGCGCGAGACGAAAGGG-3′ and 5-GGGGTCTGACGCTCAGTGGA-3′).

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: For all 55 strains, we PCR amplified and sequenced six chromosomal loci. .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen).

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: PCR to amplify a special fragment of the tissue-specific promoter with Taq or Pfu DNA polymerases (Stratagene). .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    Article Title: Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity
    Article Snippet: .. PCR reagents and T4 DNA polymerase were obtained from Invitrogen (San Diego, CA, U.S.A.). ..

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene). .. Purified plasmid DNA was sequenced by using the dideoxy termination method ( ) with Sequenase (U.S. Biochemical).

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: For bacterial expression, cDNAs were PCR-amplified using primers lacking SP sequences and directly cloned in frame with the C-terminal His tag into pET-32 Ek/LIC (Novagen). .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Selection:

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen). ..

    De-Phosphorylation Assay:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen). .. Plasmid DNA precipitation with 2 volumes of 100% cold ethanol, then wash twice with 70% ethanol Dephosphorylation of 5′-phosphorylated termini of vector DNA to prevent self-ligation using bacterial alkaline phosphatase (Invitrogen).

    cDNA Library Assay:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: Paragraph title: Generation of a Vero cell cDNA library. ... Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis.

    Shotgun Sequencing:

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: Paragraph title: Shotgun sequencing and determination of the six chromosomal Xanthomonas loci. ... Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen).

    Rapid Amplification of cDNA Ends:

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: One microgram of total cellular RNA isolated from untreated rat small intestines was reverse transcribed by using the 3′ RACE (rapid amplification of cDNA ends) System (GIBCO/BRL) according to the manufacturer’s instructions. .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene).

    Chromatin Immunoprecipitation:

    Article Title: Distinct mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine resistance revealed by transcriptome mapping in mouse striatum
    Article Snippet: .. Samples were then used to prepare the 1st strand cDNA using the One-Cycle cDNA Synthesis Kit (Affymetrix) containing SuperScript II followed by the 2nd strand cDNA synthesis with T4 DNA polymerase. cDNA was cleaned using cDNA Cleanup Spin Column (Affymetrix), and biotin-labeled cRNA was prepared using the Gene Chip IVT Labeling Kit (Affymetrix). .. Labeled cRNA was purified with Cleanup Spin Column (Affymetrix), quantified, fragmented and spiked with biotin-labeled cRNA for bioB (1,5 pM), bioC (5 pM), bioD (25 pM) and Crex (100 pM) (GeneChip® Eukaryotic Hybridization Control Kit, Affymetrix).

    Plasmid Preparation:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis. .. The adapter-ligated cDNA was recovered and ligated into BstxI-digested expression plasmid pcDNAI (Invitrogen).

    Article Title: Nucleotide Sequence of Plasmid pCNB1 from Comamonas Strain CNB-1 Reveals Novel Genetic Organization and Evolution for 4-Chloronitrobenzene Degradation ▿
    Article Snippet: .. The fragments were end blunted by T4 DNA polymerase (Fermentas) and ligated into linear vector pUC18. .. Ligated DNA was used to transform E. coli DH10B (Invitrogen), which was then plated onto LB agar plates containing ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside), and IPTG (isopropyl-β- d -thiogalactopyranoside) (Sangon).

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1). .. The Amp resistance gene fragment was amplified by PCR from the plasmid puc19 with the phosphorylated primers (5-AAACGCGCGAGACGAAAGGG-3′ and 5-GGGGTCTGACGCTCAGTGGA-3′).

    Article Title: Reduced Genetic Variation Occurs among Genes of the Highly Clonal Plant Pathogen Xanthomonas axonopodis pv. vesicatoria, Including the Effector Gene avrBs2
    Article Snippet: .. Genomic DNA was digested with MboI, and fragments of 0.5 to 1 kb were gel purified, blunted with T4 DNA polymerase, and then blunt ligated into the positive selection cloning vector pZero2.0 (Invitrogen). ..

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Paragraph title: 3.2 Plasmid Constructions ... Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene). .. Purified plasmid DNA was sequenced by using the dideoxy termination method ( ) with Sequenase (U.S. Biochemical).

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated. .. Egf1.0R51A and Egf1.0R51K were generated from the modified bacterial expression constructs by overlap extension PCR using the gene-specific primers 5′-GTAATAACAAACTATGCTAC GCG TTCCAGCAATTCTG-3′ (forward) and 5′GCTGGAA CGC GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51A ), 5′-GTAATAACAAACTATGCTAC AAG TTCCAGCAATTCTG-3′ (forward) and 5′-GCTGGAA CTT GTAGCATAGTTTGTTATTAC-3′ (reverse) (Egf1.0R51K ) (mutated Arg underlined), and vector-specific primers 5′-AGCGGATAACAATTCCCCTCTAGAAATAAT-3′ (forward) and 5′-GCTTTGTTAGCAGCCGGATCTCAGTGGT-3′ (reverse).

    Positron Emission Tomography:

    Article Title: A novel polydnavirus protein inhibits the insect prophenoloxidase activation pathway
    Article Snippet: For bacterial expression, cDNAs were PCR-amplified using primers lacking SP sequences and directly cloned in frame with the C-terminal His tag into pET-32 Ek/LIC (Novagen). .. To remove the N-terminal His tag from constructs, plasmid DNA was digested with MscI and NspV, blunt-ended with T4 DNA Polymerase (Invitrogen), and religated.

    Agarose Gel Electrophoresis:

    Article Title: Shiga Toxin Is Transported from the Endoplasmic Reticulum following Interaction with the Luminal Chaperone HEDJ/ERdj3
    Article Snippet: .. Second-strand synthesis was performed, the ends were blunted with T4 DNA polymerase, nonpalindromic BStxI adapters (Invitrogen) were ligated to the cDNA ends, and the resulting cDNA was then size selected ( > 500 bp) by agarose gel electrophoresis. .. The adapter-ligated cDNA was recovered and ligated into BstxI-digested expression plasmid pcDNAI (Invitrogen).

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Restriction endonuclease digestion of plasmid DNA (see Note 2), followed by agarose gel electrophoresis. .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    In Vitro:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water. .. In vitro transcription was performed with the MEGAscript high-yield transcription kit (Ambion) and incubated at 37° for 4 hr.

    Ethanol Precipitation:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: .. The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water. .. In vitro transcription was performed with the MEGAscript high-yield transcription kit (Ambion) and incubated at 37° for 4 hr.

    Incubation:

    Article Title: Genomewide Discovery and Classification of Candidate Ovarian Fertility Genes in the Mouse
    Article Snippet: .. The samples were blunt ended by addition of 2 μl of T4 DNA polymerase (Invitrogen) and incubation at 16° for 15 min. Ethanol precipitation was performed (70 μl 5 m NH4 OAc, 650 μl chilled 100% ethanol, 2 μl glycogen) and the sample was resuspended in 8 μl of RNase-free water. .. In vitro transcription was performed with the MEGAscript high-yield transcription kit (Ambion) and incubated at 37° for 4 hr.

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: .. A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1). .. The supernatant was concentrated and purified by Ultra-0.5 centrifugal filter devices (Amicon) again.

    Article Title: Efficient cross-species capture hybridization and next-generation sequencing of mitochondrial genomes from noninvasively sampled museum specimens
    Article Snippet: DNA extracts were blunt-ended for adapter ligation using a 1 × 20 μL master mix of 9.5 μL of H2 O, 8 μL of 5× reaction buffer for T4 DNA polymerase, 2.5 U of T4 DNA polymerase (Fermentas) in the presence of 0.4 mM dNTPs. .. Twenty microliters of the master mix was added to 20 μL of museum DNA extract and incubated for 10 min at 70°C.

    Article Title: Induction of a Rat Enteric Defensin Gene by Hemorrhagic Shock
    Article Snippet: .. PCR products were purified by glass milk adsorption (BIO101, La Jolla, Calif.), incubated in a standard fill-in reaction with T4 DNA polymerase (GIBCO/BRL), and subcloned into a BlueScript vector (Stratagene). .. Purified plasmid DNA was sequenced by using the dideoxy termination method ( ) with Sequenase (U.S. Biochemical).

    Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development
    Article Snippet: .. After the plugs solidified, they were incubated overnight at 55°C (in 100 mM Tris, pH 8.0, 25 mM EDTA, 1% sarkosyl, and 400 μg/ml proteinase K), washed in TE containing 0.5 mM PMSF for 30 min, then washed three more times in TE over 24 h. The plugs were next treated with T4 DNA polymerase by adding in a 40 μl reaction mixture containing polymerase buffer, three units T4 DNA polymerase (Life Technologies), and 100 μM dNTPs, for 1 h at 37°C. .. Plugs were then washed two more times in excess TE over 12 h and used for linker ligation.

    Colony Assay:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen). .. Transformation using MAX Efficiency DH5α Chemically Competent Cells (Invitrogen), then culture in a shaker at 37°C for 1 hour, streak out competent cells on an LB agar or agarose ampcillin plate and grow the bacterial overnight at 37°C to obtain colonies Colony screening.

    Concentration Assay:

    Article Title: High-throughput long paired-end sequencing of a Fosmid library by PacBio
    Article Snippet: A total of 200 μL DNA was end repaired with 50 units of T4 DNA polymerase (ThermoFisher), 100 units of Klenow Fragment (ThermoFisher) in 500 μL reaction mixture [10× Klenow Fragment buffer, 200 μM of dNTP Mix] and incubated at 37 °C for 1 h. The reaction mix was incubated at 65 °C for 15 min to terminate the end repairing and treated with phenol:chloroform:isoamyl alcohol (25:24:1). .. The PCR products were purified through glue recycling and concentrated by Amicon® Ultra-0.5 centrifugal filter devices to a final concentration of 0.5 mg/µL.

    DNA Purification:

    Article Title: Evaluation of promoters for use in tissue-specific gene delivery
    Article Snippet: Plasmid DNA preparations using Wizard Plus Midipreps DNA Purification System, and Maxipreps DNA purification system (Promega). .. Fill-in or removal of both 5′ or 3′ overhangs to make blunt ends using Klenow Fill-in kit (Stratagene) or T4 DNA polymerase (Invitrogen).

    Article Title: Changes in Locus-specific V(D)J Recombinase Activity Induced by Immunoglobulin Gene Products during B Cell Development
    Article Snippet: DNA purification, T4 polymerase polishing, and linker ligation were all carried out in agarose plugs, as described elsewhere (Schlissel, M.S., manuscript in preparation). .. After the plugs solidified, they were incubated overnight at 55°C (in 100 mM Tris, pH 8.0, 25 mM EDTA, 1% sarkosyl, and 400 μg/ml proteinase K), washed in TE containing 0.5 mM PMSF for 30 min, then washed three more times in TE over 24 h. The plugs were next treated with T4 DNA polymerase by adding in a 40 μl reaction mixture containing polymerase buffer, three units T4 DNA polymerase (Life Technologies), and 100 μM dNTPs, for 1 h at 37°C.

    Gel Purification:

    Article Title: Molecular Cloning, Sequence Analysis, and Heterologous Expression of the Phosphinothricin Tripeptide Biosynthetic Gene Cluster from Streptomyces viridochromogenes DSM 40736
    Article Snippet: Endonucleases, T4 DNA polymerase, and T4 DNA ligase were purchased from Invitrogen (Carlsbad, Calif.) and New England Biolabs (Beverly, Mass.). .. DNA fragments for cloning were isolated after gel purification with the Agarace enzyme (Promega, Madison, Wis.) according to the manufacturer's recommendations.

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