thermo fisher scientific t4 polymerase  (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
    Applications:
    Cloning|PCR Cloning|Restriction Enzyme Cloning|Mutagenesis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher thermo fisher scientific t4 polymerase
    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
    https://www.bioz.com/result/thermo fisher scientific t4 polymerase/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    t4 dna ligase

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    Polymerase Chain Reaction:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Incubation:

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

    Expressing:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Plasmid Preparation:

    Article Title: Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice
    Article Snippet: .. The PCR products were ligated into the Bam HI/ Not I site of pPK-CMV-F4 (PromoCell GmbH, Heidelberg, Germany) mammalian expression vector using T4 ligase (Invitrogen, Carlsbad, CA, USA). .. All plasmids containing correct inserts (as confirmed by DNA sequencing) were propagated in Escherichia coli NOVA XG cells, amplified, extracted and purified using conventional methods.

    Ligation:

    Article Title: Golden GATEway Cloning - A Combinatorial Approach to Generate Fusion and Recombination Constructs
    Article Snippet: .. The 10nM annealed double-stranded oligo dilution was used as an insert in a standard ligation reaction with T4 ligase (5U, Thermo, Fisher) .. Golden Gate Protocol Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O.

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection
    Article Snippet: .. Equal molar amounts (typically 250–500 ng at ∼ 100 – 250 ng/µl ) of orthogonally protected synthons were mixed, 0.5–1 unit T4 ligase (Fermentas) and T4 ligase buffer (Fermentas) were added and the ligation mixture was incubated for 10–20 min at 16°C. .. Adding additional ligase had little effect on ligation efficiency.

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  • 99
    Thermo Fisher dnase i
    Genetic organization of the  cer  cluster with predicted promoters and RT-PCR analysis of  cer  locus. (A)  cer  genes are indicated by differently colored arrows: the precursor genes  cerA1  to - A7  are indicated by green arrows, the modification enzyme gene  cerM  in blue, the orphan regulator gene  cerR  in yellow, the protease and transporter genes  cerT  and  cerP  in purple, and the immunity genes  cerF  and  cerE  in dark red. The gray arrows indicate quorum-sensing component genes  comQXPA . P cerA  is the predicted promoter of  cerA , P cerR  is the predicted promoter of  cerR , and P cerF  is the predicted promoter of  cerF . (B) RT-PCR analysis of the  cer  locus. RNA was extracted from the WT strain and the BceR strain at 8 h after inoculation. RT-PCR amplification of 16S rRNA and intergenic regions between  cerA  and  cerM ,  cerR  and  cerT ,  cerP  and  cerF , and  cerF  and  cerE . M, molecular standard; G, positive controls with genomic DNA; S, cDNA from the RNA sample; CK, negative controls consisting of DNase I-treated RNA sample.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    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 165 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 polynucleotide kinase
    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with <t>T4</t> polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).
    T4 Polynucleotide Kinase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Genetic organization of the  cer  cluster with predicted promoters and RT-PCR analysis of  cer  locus. (A)  cer  genes are indicated by differently colored arrows: the precursor genes  cerA1  to - A7  are indicated by green arrows, the modification enzyme gene  cerM  in blue, the orphan regulator gene  cerR  in yellow, the protease and transporter genes  cerT  and  cerP  in purple, and the immunity genes  cerF  and  cerE  in dark red. The gray arrows indicate quorum-sensing component genes  comQXPA . P cerA  is the predicted promoter of  cerA , P cerR  is the predicted promoter of  cerR , and P cerF  is the predicted promoter of  cerF . (B) RT-PCR analysis of the  cer  locus. RNA was extracted from the WT strain and the BceR strain at 8 h after inoculation. RT-PCR amplification of 16S rRNA and intergenic regions between  cerA  and  cerM ,  cerR  and  cerT ,  cerP  and  cerF , and  cerF  and  cerE . M, molecular standard; G, positive controls with genomic DNA; S, cDNA from the RNA sample; CK, negative controls consisting of DNase I-treated RNA sample.

    Journal: Applied and Environmental Microbiology

    Article Title: CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus

    doi: 10.1128/AEM.02245-17

    Figure Lengend Snippet: Genetic organization of the cer cluster with predicted promoters and RT-PCR analysis of cer locus. (A) cer genes are indicated by differently colored arrows: the precursor genes cerA1 to - A7 are indicated by green arrows, the modification enzyme gene cerM in blue, the orphan regulator gene cerR in yellow, the protease and transporter genes cerT and cerP in purple, and the immunity genes cerF and cerE in dark red. The gray arrows indicate quorum-sensing component genes comQXPA . P cerA is the predicted promoter of cerA , P cerR is the predicted promoter of cerR , and P cerF is the predicted promoter of cerF . (B) RT-PCR analysis of the cer locus. RNA was extracted from the WT strain and the BceR strain at 8 h after inoculation. RT-PCR amplification of 16S rRNA and intergenic regions between cerA and cerM , cerR and cerT , cerP and cerF , and cerF and cerE . M, molecular standard; G, positive controls with genomic DNA; S, cDNA from the RNA sample; CK, negative controls consisting of DNase I-treated RNA sample.

    Article Snippet: After being purified from the agarose gel, the probes (200 ng) and proteins at different concentrations were added to a final reaction volume of 50 μl and incubated at 25°C for 30 min. DNase I (Thermo Scientific, USA) digestions were carried out for 30 s at 37°C and stopped with EDTA.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Modification, Amplification

    Binding site analysis of CerR on different target regions. (A and B) DNase I footprinting for determination of CerR binding sites on P cerA (A) and P cerR (B). Each line represents 200 ng DNA probes and corresponding gradient concentrations of His 6 -CerR (0, 0.1, 0.5, and 1.0 μM). The traces indicate the signal strengths of different length of DNA sequences. The nucleotide sequences corresponding to the protected fragments are listed at the bottom. (C) The predicted binding site (site I) of CerR on P cerA . (D) The predicted binding site (site II) of CerR on P cerR . The translation start codons are marked by red, and the sequences protected by CerR are underlined and in bold italics.

    Journal: Applied and Environmental Microbiology

    Article Title: CerR, a Single-Domain Regulatory Protein of the LuxR Family, Promotes Cerecidin Production and Immunity in Bacillus cereus

    doi: 10.1128/AEM.02245-17

    Figure Lengend Snippet: Binding site analysis of CerR on different target regions. (A and B) DNase I footprinting for determination of CerR binding sites on P cerA (A) and P cerR (B). Each line represents 200 ng DNA probes and corresponding gradient concentrations of His 6 -CerR (0, 0.1, 0.5, and 1.0 μM). The traces indicate the signal strengths of different length of DNA sequences. The nucleotide sequences corresponding to the protected fragments are listed at the bottom. (C) The predicted binding site (site I) of CerR on P cerA . (D) The predicted binding site (site II) of CerR on P cerR . The translation start codons are marked by red, and the sequences protected by CerR are underlined and in bold italics.

    Article Snippet: After being purified from the agarose gel, the probes (200 ng) and proteins at different concentrations were added to a final reaction volume of 50 μl and incubated at 25°C for 30 min. DNase I (Thermo Scientific, USA) digestions were carried out for 30 s at 37°C and stopped with EDTA.

    Techniques: Binding Assay, Footprinting

    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: 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).

    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: 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).

    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: 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).

    Techniques: Ligation, Sequencing, Ancient DNA Assay

    ΔFANCJ cells accumulate ssDNA gaps due to their inability to replicate past DNA sequence–dependent fork barriers. (A) Dot plot of fork speed of individual fibers from WT and ΔFANCJ cells either untreated or treated with 5 µM telomestatin for 24 h before subsequent labeling with IdU and CldU. Only continuously replicating red-green fibers were measured ( n ≥ 300). (B) Dot plot of CldU tract length ratios of associated sister forks from untreated or telomestatin-treated WT and ΔFANCJ cells ( n ≥ 50). (C) A schematic diagram of the gap-labeling procedure using T4 DNA polymerase. (D) DT40 cells were incubated with telomestatin for different times and genomic DNA was subsequently isolated and used as a template for the gap-filling assay using T4 DNA polymerase. 32 P-labeled DNA samples were resolved on an alkaline agarose gel, transferred onto a membrane, and scanned by a phosphorimager. Total genomic DNA run on a nondenaturing gel is shown below for quantification purposes. (E) WT and ΔFANCJ cells were treated with different doses of telomestatin for 24 h. The labeled nascent DNA extended by T4 polymerase was separated on a denaturing gel.

    Journal: The Journal of Cell Biology

    Article Title: FANCJ couples replication past natural fork barriers with maintenance of chromatin structure

    doi: 10.1083/jcb.201208009

    Figure Lengend Snippet: ΔFANCJ cells accumulate ssDNA gaps due to their inability to replicate past DNA sequence–dependent fork barriers. (A) Dot plot of fork speed of individual fibers from WT and ΔFANCJ cells either untreated or treated with 5 µM telomestatin for 24 h before subsequent labeling with IdU and CldU. Only continuously replicating red-green fibers were measured ( n ≥ 300). (B) Dot plot of CldU tract length ratios of associated sister forks from untreated or telomestatin-treated WT and ΔFANCJ cells ( n ≥ 50). (C) A schematic diagram of the gap-labeling procedure using T4 DNA polymerase. (D) DT40 cells were incubated with telomestatin for different times and genomic DNA was subsequently isolated and used as a template for the gap-filling assay using T4 DNA polymerase. 32 P-labeled DNA samples were resolved on an alkaline agarose gel, transferred onto a membrane, and scanned by a phosphorimager. Total genomic DNA run on a nondenaturing gel is shown below for quantification purposes. (E) WT and ΔFANCJ cells were treated with different doses of telomestatin for 24 h. The labeled nascent DNA extended by T4 polymerase was separated on a denaturing gel.

    Article Snippet: Gap assay 1.5 µg of genomic DNA was incubated with 7.5 units of T4 DNA polymerase (Thermo Fisher Scientific), dNTPs, and α-[32 P]ATP at 37°C in a buffer containing 67 mM Tris-HCl, pH 8.8, 6.6 mM MgCl2 , 1 mM DTT, and 16.8 mM (NH4 )2 SO4 .

    Techniques: Sequencing, Labeling, Incubation, Isolation, Agarose Gel Electrophoresis

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Techniques: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis