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    Thermo Fisher thermo fisher scientific t4 polymerase
    Thermo Fisher Scientific T4 Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 1 article reviews
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    92
    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: 92/100, based on 2326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/Thermo Fisher
    Average 92 stars, based on 2326 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2020-05
    92/100 stars
      Buy from Supplier

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    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

    J κ 1 and J H 2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for J H 2 (A) or J κ 1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32 P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for J H 2 and +4 for J κ 1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: J κ 1 and J H 2 coding ends have 3′ overhangs. (A and B) DNA purified in agarose plugs from newborn-mouse thymocytes (A) or 103 bcl2/4 cells cultured at 33 or 39°C (B) (shift − or +) was ligated to the BW linker without pretreatment (lanes none) or after treatment with either T4 DNA polymerase (lanes T4 Pol) or mung bean nuclease (lanes MB-N). Ligated plugs were analyzed by PCR for J H 2 (A) or J κ 1 (B) coding-end (ce) breaks (arrows). The lanes labeled 63-12 were control LM-PCR assays with 63-12 cell DNA. Lanes 1 and 2 in panel A represent independent thymocyte DNA samples. (C) Amplified products from lanes 1, 3, and 4 in panel A and lanes 2, 4, and 6 in panel B were gel purified, reamplified for five cycles with a 32 P-labeled specific oligonucleotide, and analyzed by denaturing polyacrylamide gel electrophoresis. In each case, the arrow indicates the predominant broken-ended molecule (+9 for J H 2 and +4 for J κ 1) and the tick marks indicate 1-nt intervals (determined by the electrophoresis of a DNA sequencing reaction mixture on the same gel). Lanes: N, no pretreatment; T, T4 DNA polymerase pretreatment; M, mung bean nuclease pretreatment.

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Purification, Cell Culture, Polymerase Chain Reaction, Labeling, Amplification, Polyacrylamide Gel Electrophoresis, Electrophoresis, DNA Sequencing

    V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: V(D)J recombination reaction pathway and LM-PCR assay for reaction intermediates. (A) Diagram of the reactants (top), broken DNA intermediates (middle), and products (bottom) of V(D)J recombination. V and J gene segments, with their associated RSSs (heptamer [H] and nonamer [N]) are recognized and cleaved by the recombinase at the RSS–coding-segment junction (arrow), generating coding-end and signal-end fragments. These ends are joined to form a coding joint and a signal joint. (B) LM-PCR assay for broken-ended recombination reaction intermediates. The BW linker is ligated to available ends in total genomic DNA by using T4 DNA ligase. The sites of linker ligation are revealed by a set of nested PCR assays with a linker primer (BW-1) and locus-specific primers (open arrows labeled 1, 2, 4, and 5). Blots of PCR products were probed with internal oligonucleotides (solid lines labeled 3 and 6).

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Polymerase Chain Reaction, Ligation, Nested PCR, Labeling

    T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken J κ 1 coding (A), J κ 1 and J κ 2 signal (B), and J H 2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: T4 DNA polymerase treatment enhances LM-PCR detection of broken coding ends. (A to C) DNA samples prepared by the agarose plug method from uninduced (33°C [33 degr]) and induced (39°C [39 degr]) 103 bcl2/4 cells (A and B) and from newborn thymus (C) were analyzed by LM-PCR for broken J κ 1 coding (A), J κ 1 and J κ 2 signal (B), and J H 2 coding and signal (C) ends without (lanes −) or with (lanes +) T4 DNA polymerase (T4 pol) pretreatment. Controls included identically prepared and treated 63-12 (RAG-2-deficient) cell DNA and buffer (lane C). DNA samples from panel C were amplified with primers specific for a nonrearranging genomic locus, demonstrating the presence of DNA in all samples. (D) Ethidium bromide-stained agarose gel of these control amplifications. Lanes 1 to 4 correspond to samples 1 to 4 in panel C, lanes 5 to 8 correspond to samples 6 to 9 in panel C, and lane 9 is a buffer-only control amplification. Lanes 1 to 6 of panels A and B were shown to contain equivalent amounts of DNA by a similar method (data not shown). ce, coding ends; se, signal ends.

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Polymerase Chain Reaction, Amplification, Staining, Agarose Gel Electrophoresis

    Length heterogeneity of amplified coding ends and joints. D H , J H 1, J H 2, V κ , and J κ 1 coding ends (ce) (A, B, C, E, and F) and DJ H and VJ κ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ- 32 P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJ H and VJ κ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact V κ genes (see the text).

    Journal: Molecular and Cellular Biology

    Article Title: Structure of Nonhairpin Coding-End DNA Breaks in Cells Undergoing V(D)J Recombination

    doi:

    Figure Lengend Snippet: Length heterogeneity of amplified coding ends and joints. D H , J H 1, J H 2, V κ , and J κ 1 coding ends (ce) (A, B, C, E, and F) and DJ H and VJ κ joints (D and G) were amplified from T4 polymerase-treated thymus or induced 103 bcl2/4 cell DNA. The amplified fragments were gel purified and end labeled with [γ- 32 P]ATP by using T4 DNA kinase. Labeled fragments were electrophoresed on 6% denaturing polyacrylamide gels alongside DNA sequencing ladders used as size markers. The diagrams adjacent to each coding-fragment gel image indicate the sequence position based on these comigrating sequence markers. Position zero in the diagrams corresponds to the full-length coding end (i.e., the junction between the RSS and the coding segment), positive numbers indicate coding-end deletions, and negative numbers indicate longer-than-full-length coding-end lengths. The diagrams adjoining DJ H and VJ κ joints indicate successive nucleotide lengths. In panel E, the lane labeled Vκ mkr contains a radiolabeled amplification product of genomic DNA demonstrating the length heterogeneity of intact V κ genes (see the text).

    Article Snippet: Some plug DNA samples were subjected to T4 DNA polymerase treatment by incubating 40 μl of plug in an 80-μl reaction mixture with manufacturer’s buffer (Life Technologies), 5 U of T4 DNA polymerase, and 100 μM deoxynucleoside triphosphates at 37°C for 1 h. Other plugs were treated with various amounts of mung bean nuclease (BRL) under conditions previously reported by Zhu and Roth ( ).

    Techniques: Amplification, Purification, Labeling, DNA Sequencing, Sequencing

    UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Journal: The Journal of Biological Chemistry

    Article Title: UV Damage in DNA Promotes Nucleosome Unwrapping *

    doi: 10.1074/jbc.M110.140087

    Figure Lengend Snippet: UV damage does not affect NCP reconstitution with the 601 sequence. A , NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 m m . The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B , presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32 P-labeled DNA to show equal loading. C , Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase ( pol ) digestion. The digested samples were blotted on the nylon membrane and probed with with 32 P-labeled DNA. D , quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars .

    Article Snippet: Briefly, samples were incubated with 2.5 units of T4 DNA polymerase-exonuclease (Fermentas) at 37 °C for 2 h. The reaction was stopped by heating at 65 °C for 10 min.

    Techniques: Sequencing, Labeling, Concentration Assay, Staining, Southern Blot, Irradiation

    Validation of Kir isoform typing with reporter enzyme ScrF I. Degenerate PCR with sub-family centred primers was performed on Kir subclone templates and products were T4 DNA polymerase end-labelled according to Materials and Methods . Diagnostic restriction digests were performed using ScrF I and an isoform specific duplex banding pattern generated consistent with the predicted banding patterns described in Figure 1 . Faint low MW fragments (

    Journal: BMC Genomics

    Article Title: Multigene family isoform profiling from blood cell lineages

    doi: 10.1186/1471-2164-3-22

    Figure Lengend Snippet: Validation of Kir isoform typing with reporter enzyme ScrF I. Degenerate PCR with sub-family centred primers was performed on Kir subclone templates and products were T4 DNA polymerase end-labelled according to Materials and Methods . Diagnostic restriction digests were performed using ScrF I and an isoform specific duplex banding pattern generated consistent with the predicted banding patterns described in Figure 1 . Faint low MW fragments (

    Article Snippet: T4 DNA Polymerase end-labelling of degenerate PCR products The products from the degenerate, sub-family centred PCR were excised and purified from agarose gels using a kit (Hybaid Ltd, Middlesex, UK) according to manufacturers protocol, and eluted into 20 μl elution solution.

    Techniques: Polymerase Chain Reaction, Diagnostic Assay, Generated