t4 ligase  (Thermo Fisher)


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    Name:
    T4 DNA Polymerase 5 U µL
    Description:
    Thermo Scientific T4 DNA Polymerase is a template dependent DNA polymerase that catalyzes 5 3 synthesis from primed single stranded DNA The enzyme has a 3 5 exonuclease activity but lacks 5 3 exonuclease activity Highlights• Stronger 3 5 exonuclease activity on single stranded than on double stranded DNA and greater more than 200 times than DNA polymerase I E coli and Klenow fragment• Active in Thermo Scientific restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• Blunting of DNA ends fill in of 5 overhangs or and removal of 3 overhangs see​ References1 2 • Blunting of PCR products with 3 dA overhangs• Synthesis of labeled DNA probes by the replacement reaction see​ Reference3 • Oligonucleotide directed site specific mutagenesis see​ Reference4 • Ligation independent cloning of PCR products
    Catalog Number:
    EP0061
    Price:
    None
    Category:
    Proteins Enzymes Peptides
    Applications:
    Cloning|PCR Cloning|Restriction Enzyme Cloning|Mutagenesis
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    Structured Review

    Thermo Fisher t4 ligase
    Thermo Scientific T4 DNA Polymerase is a template dependent DNA polymerase that catalyzes 5 3 synthesis from primed single stranded DNA The enzyme has a 3 5 exonuclease activity but lacks 5 3 exonuclease activity Highlights• Stronger 3 5 exonuclease activity on single stranded than on double stranded DNA and greater more than 200 times than DNA polymerase I E coli and Klenow fragment• Active in Thermo Scientific restriction enzyme PCR RT and T4 DNA Ligase buffersApplications• Blunting of DNA ends fill in of 5 overhangs or and removal of 3 overhangs see​ References1 2 • Blunting of PCR products with 3 dA overhangs• Synthesis of labeled DNA probes by the replacement reaction see​ Reference3 • Oligonucleotide directed site specific mutagenesis see​ Reference4 • Ligation independent cloning of PCR products
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    Average 99 stars, based on 1 article reviews
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    Related Articles

    Plasmid Preparation:

    Article Title: Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase.
    Article Snippet: The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. .. The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. .. The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified.

    Construct:

    Article Title: Biotransformation of L-lysine to L-pipecolic acid catalyzed by L-lysine 6-aminotransferase and pyrroline-5-carboxylate reductase.
    Article Snippet: The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. .. The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified. .. The enzyme involved in the reduction of delta1-piperideine-6-carboxylate (P6C) to L-pipecolic acid (L-PA) has never been identified.

    Activity Assay:

    Article Title: Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
    Article Snippet: .. Considering that the T4 pol is exerting its exonuclease activity on both ends, the overall T4 pol exonuclease rate would be approximately half this figure, at 1.6 bases/s or 96 bases/min/molecule end. .. To further appreciate the effects of T4 pol exonuclease activity on different sized products, we observed the effects of T4 pol together with a 100 bp ladder, treated for 30 s, 1 min, 3 min and 5 min.

    Ligation:

    Article Title: Herbaspirillum seropedicae Differentially Expressed Genes in Response to Iron Availability
    Article Snippet: Double and triple mutants generated in this work were constructed by successive matings in the appropriate parental strains with the appropriate plasmids carried in E. coli TOP10 donors and a E. coli TOP10 pRK2013 as a helper strain. .. Restriction enzymes were purchased from Promega, and ligation reactions were performed using T4 ligase (Invitrogen). .. Plasmid preparation by alkaline lysis, preparation of competent cells, and transformation by heat shock were performed by standard protocols ( ).

    Modification:

    Article Title: UhAVR1, an HR-Triggering Avirulence Effector of Ustilago hordei, Is Secreted via the ER–Golgi Pathway, Localizes to the Cytosol of Barley Cells during in Planta-Expression, and Contributes to Virulence Early in Infection
    Article Snippet: Since two UmHsp70 promoter sequences in the same construct may cause instability, the UmHsp70 hygromycin B resistance cassette needed to be replaced. .. Therefore, pCM60 was modified to delete the hygromycin cassette through Nar1 digestion, blunt-end formation by the Klenow fragment of DNA polymerase I, a second digestion with Sph1 and blunt-end formation by T4 polymerase, and self-ligation. .. This interim vector was subsequently digested with PvuII and Xma1, after which a 1.9 kb Xma1-EcoRV fragment from plasmid pDONR-CbxR, harbouring the carboxin resistance gene [ ], was inserted to give plasmid pCM100 a unique BamHI site.

    Generated:

    Article Title: An Overview of Genomics, Phylogenomics and Proteomics Approaches in Ascomycota
    Article Snippet: A voltage bias generated at the two sides of a membrane produces current which drives ssDNA (or RNA) through the nanopore. .. An enzyme (polymerase or helicase) is bound to the nucleic acid and works as a ratchet, controlling the step-wise passage of the nucleotides, allowing the detection of the different current perturbation (few pA) generated by each nucleotide when passing through the narrowest part of the nanopore [ ]. .. Reads length limit is mostly determined by DNA fragments length and ultra-long reads, up to almost 1 Mb, were obtained [ ]; accuracy can be low (85%, increased to 97% when sequencing both strand) and systematic errors occur in homopolymers [ ].

    Fluorescence:

    Article Title: Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
    Article Snippet: We assessed this in two ways, i.e. measurement of the ‘rate of decay’ of Sybr fluorescence, and, electrophoretic mobility shift. .. For Sybr fluorescence, we tested this by using 100 ng of a purified 762 bp PCR product (gfp ) together with 1 U T4 pol (per reaction) and incubating the samples at 37 °C. .. At the desired time-points, individual reactions were stopped with the addition of EDTA and Sybr Green I, a double-stranded DNA (dsDNA) stain, was added.

    Purification:

    Article Title: Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
    Article Snippet: We assessed this in two ways, i.e. measurement of the ‘rate of decay’ of Sybr fluorescence, and, electrophoretic mobility shift. .. For Sybr fluorescence, we tested this by using 100 ng of a purified 762 bp PCR product (gfp ) together with 1 U T4 pol (per reaction) and incubating the samples at 37 °C. .. At the desired time-points, individual reactions were stopped with the addition of EDTA and Sybr Green I, a double-stranded DNA (dsDNA) stain, was added.

    Polymerase Chain Reaction:

    Article Title: Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method
    Article Snippet: We assessed this in two ways, i.e. measurement of the ‘rate of decay’ of Sybr fluorescence, and, electrophoretic mobility shift. .. For Sybr fluorescence, we tested this by using 100 ng of a purified 762 bp PCR product (gfp ) together with 1 U T4 pol (per reaction) and incubating the samples at 37 °C. .. At the desired time-points, individual reactions were stopped with the addition of EDTA and Sybr Green I, a double-stranded DNA (dsDNA) stain, was added.

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    Thermo Fisher t4 dna ligase
    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with <t>T4</t> DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
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    98
    Thermo Fisher t4 rna ligase
    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with <t>T4</t> RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    doi: 10.1016/j.bpj.2018.08.013

    Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Article Snippet: Immobilized target DNA was incubated with 50 nM of each upstream and 50 nM of each downstream barcode (independent of the number of different barcode sequences used) and 14 Weiss units/mL of T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA) in freshly prepared ligation buffer (40 mM Tris-HCl (pH 7.6), 10 mM MgCl2 , 10 mM dithiothreitol, 0.5 mM ATP) for 1 h at 25°C.

    Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkx033

    Figure Lengend 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.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkx033

    Figure Lengend 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.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: 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.

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkx033

    Figure Lengend 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.

    Article Snippet: Ligation was performed in 80 μl reactions containing 1 × T4 RNA ligase buffer, 20% PEG-8000, 0.5 mM ATP, 10/20 pmol of adapter splinter mix CL78/TL38, 1, 2 or 4 pmol acceptor oligonucleotide and 30 U T4 DNA ligase (ThermoFisher Scientific).

    Techniques: Ligation, Sequencing, Ancient DNA Assay

    AWH-induced IgG1 production is associated with an increase in the number of IgG1-switched cells. Genomic DNA was isolated from either the TSI-18 and IB4 hybridomas (A) or the spleen cells of mice treated with either AWH, worms ( Nb ), or FIA (B) as described in Materials and Methods. The DNA was digested with Eco RI, ligated with T4 DNA ligase, and amplified by PCR using primers specific for the recombined switch regions. nAChRe levels in all samples were also determined by DC-PCR to control for equal template loading and allow semiquantitation (comparison) of the Sμ-Sγ1 product. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of six experiments. (A) Lane 1, TSI-18 (IgG1-producing hybridoma); lane 2, IB4 (IgG2a-producing hybridoma); lane 3, no DNA (control for PCR contamination); lane 4, TSI-18 (nAChRe amplicon from IgG1-producing hybridoma).

    Journal: Infection and Immunity

    Article Title: Extract of Nippostrongylus brasiliensis Stimulates Polyclonal Type-2 Immunoglobulin Response by Inducing De Novo Class Switch

    doi:

    Figure Lengend Snippet: AWH-induced IgG1 production is associated with an increase in the number of IgG1-switched cells. Genomic DNA was isolated from either the TSI-18 and IB4 hybridomas (A) or the spleen cells of mice treated with either AWH, worms ( Nb ), or FIA (B) as described in Materials and Methods. The DNA was digested with Eco RI, ligated with T4 DNA ligase, and amplified by PCR using primers specific for the recombined switch regions. nAChRe levels in all samples were also determined by DC-PCR to control for equal template loading and allow semiquantitation (comparison) of the Sμ-Sγ1 product. PCR amplicons were resolved on a 1.5% agarose gel with ethidium bromide staining. Results are representative of six experiments. (A) Lane 1, TSI-18 (IgG1-producing hybridoma); lane 2, IB4 (IgG2a-producing hybridoma); lane 3, no DNA (control for PCR contamination); lane 4, TSI-18 (nAChRe amplicon from IgG1-producing hybridoma).

    Article Snippet: The reaction mixtures were then incubated overnight in a 37°C waterbath, following which the enzyme was inactivated by incubation at 70°C for 20 min. For ligation (circularization), 10 to 20 μl of digested DNA samples was placed in 1.5-ml microcentrifuge tubes to which 20 μl of 5× T4 DNA ligase buffer (1× final; Life Technologies), 2 μl (20 U) of T4 DNA ligase (Life Technologies), and double-distilled water were added to a final volume of 100 μl.

    Techniques: Isolation, Mouse Assay, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

    Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

    Journal: RNA

    Article Title: Stable PNPase RNAi silencing: Its effect on the processing and adenylation of human mitochondrial RNA

    doi: 10.1261/rna.697308

    Figure Lengend Snippet: Improper polyadenylation and processing of the COX1 transcript in PNPase-silenced cell lines. ( A ) cRT-PCR labeling and sequencing methods, used to investigate the 5′ and 3′ ends of a target mRNA, are described. Both start with the circularization of total RNA which contains the target mRNA, with T4 RNA ligase. Next, a gene-specific reverse oligo, generally termed R1, is used to prime reverse transcription, initiated ∼100 nt downstream of the 5′ end. Afterward, two consecutive PCR reactions with F1+R2 and F2+R2 oligos, respectively, amplify the adjoined 5′ and 3′ extremities and simultaneously increase specificity. At this point, there are two options: For sequencing, the products are cloned to T/A vectors, PCR-screened, and sequenced, in order to analyze individual clone sequences (cRT-PCR sequencing). To obtain a more global view of the target mRNA population instead, a third PCR reaction, similar to the second, can be applied, in which either the R2 or F2 oligo is labeled with [γ- 32 P]ATP. Products are resolved in 10% acrylamide gel, followed by autoradiography (cRT-PCR labeling). The 3′ poly(A) tail lengths can be calculated by subtracting the expected length of a properly processed naked 3′ end molecule from that of the actual product as compared to a nucleotide ladder. ( B ) The 3′ and 5′ ends of COX1 were analyzed in control (wt and EV) and PNPase-silenced (E1, E3, and G3) cells using the cRT-PCR labeling technique (as described above for A ). Products were resolved by 10% denaturing PAGE, followed by autoradiography, and product size was determined by comparison to a nucleotide ladder produced by alkaline hydrolysis of a [ 32 P]RNA (lane M ). Assuming proper processing of the mRNA, the product size represents the length of the poly(A) tail added to the 3′ end, a naked 3′ end marked as “0.” However, products could also originate from molecules with impaired processing. In order to differentiate between these two possibilities, cRT-PCR sequencing was performed as shown in part C of the figure. ( C ) cRT-PCR sequencing of COX1 is shown. The region of the human mitochondrial genome containing the COX1 gene is schematically displayed at the bottom . The first nucleotide of the COX1 transcript at the 5′ end is marked as +1. The translation initiation codon starts at number +4, and the amino acid coding region is colored in dark gray with the two diagonal lines indicating that it is not drawn to scale. The 5′ and 3′ UTRs, composed of 3 nt and the tRNA K antisense, respectively, are shown in light gray. The flanking sequences, including the 9-nt intergenic region and tRNA Y antisense located upstream of the COX1 gene, are marked with a dashed white line. Four black arrows represent the R2, R1, F2, and F1 primers used in cRT-PCR. Above the gene scheme, individually sequenced COX1 clones are shown for each cell line. A dashed line symbols the inferred internal part of the COX1 mRNA that was not physically isolated, as only the transcript extremities were amplified (as described above for A ). Black lines show the sequenced segments of the 5′ and 3′ ends with the relative position aligned to the scheme below. The 5′ end sites, initiating at positions other than the proper +1, are labeled in parentheses. At the 3′ end of the transcript, either the number of adenosines is indicated or, in parentheses, the post-transcriptionally added nonadenosine extensions that could be located either at the 3′ or at the 5′ end of the transcript.

    Article Snippet: Circular reverse transcription PCR (cRT-PCR) labeling and sequencing were used to determine both 5′ and 3′ extremities of various mtRNA as described ( ; ; ) Briefly, 5 μg of total RNA was circularized with T4 RNA ligase (Fermentas), followed by phenol–chloroform extraction and ethanol precipitation. cDNA was synthesized using a gene-specific reverse primer, generally termed R1, and StrataScript 5.0 (Stratagene) and used as a template for a PCR reaction, primed with a nested reverse primer, R2, and a forward primer, F1.

    Techniques: Polymerase Chain Reaction, Labeling, Sequencing, Clone Assay, Acrylamide Gel Assay, Autoradiography, Polyacrylamide Gel Electrophoresis, Produced, Isolation, Amplification