t4 dna Search Results


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  • 95
    New England Biolabs t4 dna polymerase
    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of <t>T4</t> DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 6750 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore t4 dna ligase
    HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of <t>T4</t> DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).
    T4 Dna Ligase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 473 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 dna ligase
    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and <t>T4</t> DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 19342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 dna polymerase exonuclease
    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 <t>T4</t> 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 .
    T4 Dna Polymerase Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa t4 dna ligase
    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by <t>T4</t> DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.
    T4 Dna Ligase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 5426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa t4 dna polymerase
    The PCR product of a foreign gene was amplified by <t>T4</t> DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
    T4 Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 730 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore t4 dna polymerase
    LIC procedure using pMCSG vectors. All MCSG vectors contain an Ssp I site (AATATT) positioned immediately after the sequence encoding the TEV protease recognition site. Cleavage with Ssp I (a blunt cutter) followed by treatment with <t>T4</t> DNA polymerase in
    T4 Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 702 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs phage t4 dna ligase
    Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with <t>T4</t> DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.
    Phage T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    TaKaRa t4 dna
    Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with <t>T4</t> DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.
    T4 Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    FUJIFILM t4 dna
    (a) AFM scan and SEM image (insert) of the UV curable resin stamp possessing a positive tone of the 2-D nanochannels. Channels were imprinted into PMMA with dimensions (width × depth) of nc 1 = 300 × 200 nm, nc 2 = 250 × 155 nm, nc 3 = 190 × 95 nm, nc 4 = 150 × 60 nm and nc 5 = 110 × 25 nm. (b) Bar graphs showing the signal-to-noise ratio (SNR) at 2 s exposure time for the devices with untreated PMMA substrate enclosed with a plasma treated COC cover plate, U-PMMA/(PL-COC), and plasma treated substrate enclosed with a plasma treated PMMA cover plate, PL-PMMA/(PL-PMMA) filled with a 5 mM FITC solution. The error bars represent the standard deviation in measurements from ten separate devices. Insert shows unprocessed images of the seeding test for U-PMMA/(PL-COC). (c) Unprocessed representative frames of <t>T4</t> DNA molecules elongated in enclosed nanochannels for the hybrid devices. Images were acquired at 10 ms exposure time with the driving field turned-off. Note that nc 6 = 35 × 35 nm. (d) Log-log plot showing the T4 DNA extension as a function of the geometric average depth of the nanochannels. The DNA extension was normalized to a total contour length (L c ) of 64 µm for the dye-labeled molecules. The red and blue dashed lines are the deGennes and Odijk predictions, respectively. The black solid line is the best power-law fit to the data points obtained from the nanochannels with an average geometric depth range of 53 nm to 200 nm.
    T4 Dna, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher t4 dna ligation buffer
    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using <t>T4</t> DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.
    T4 Dna Ligation Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher t4 ligase buffer
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Ligase Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Beijing CWBio t4 dna ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Dna Ligase, supplied by Beijing CWBio, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Bio-Rad t4 dna ligase
    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of <t>T4</t> ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.
    T4 Dna Ligase, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 97/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Mannheim t4 dna ligase
    <t>T4</t> DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.
    T4 Dna Ligase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 885 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim t4 dna polymerase
    Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) <t>T4</t> DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.
    T4 Dna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Enzymatics t4 dna ligase
    3′ Branch ligation by <t>T4</t> DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.
    T4 Dna Ligase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 97/100, based on 648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Enzymatics t4 dna polymerase
    3′ Branch ligation by <t>T4</t> DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.
    T4 Dna Polymerase, supplied by Enzymatics, used in various techniques. Bioz Stars score: 94/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Fisher Scientific t4 dna ligase
    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by <t>T4</t> DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.
    T4 Dna Ligase, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 97/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare t4 dna ligase
    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or <t>T4</t> DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.
    T4 Dna Ligase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc t4 dna polymerase
    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or <t>T4</t> DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.
    T4 Dna Polymerase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Nanocs t4 dna ligase
    Optimization of D-probe complementary sequence length. (A) Complementary sequences of four probe sets for miR-143. (B) The fluorescence intensities of D-probes bound to C-probes in the presence of <t>T4</t> DNA ligase. Data represent the mean ± S.E. (n = 3). (C) Correlation between input miR-143 and the signal of D-probe-143-(8) in the presence (filled circle) or absence (open circle) of T4 DNA ligase. The upper axis indicates the final concentration of input miR-143 in the hybridization chamber while the lower axis indicates the total amount of input miR-143. Data in the linear range were fitted by a linear expression (gray lines). Data represent the mean ± S.E. (n = 3).
    T4 Dna Ligase, supplied by Nanocs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Promega t4 dna ligase
    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with <t>T4</t> DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.
    T4 Dna Ligase, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 8631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega t4 dna polymerase
    Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with <t>T4</t> DNA polymerase, which robustly
    T4 Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 940 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Roche t4 dna ligase
    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using <t>T4</t> DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).
    T4 Dna Ligase, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 3990 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche t4 dna polymerase
    Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with <t>T4</t> DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.
    T4 Dna Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 445 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech t4 dna ligase
    Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with <t>T4</t> DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.
    T4 Dna Ligase, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 97/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Shanghai Genechem t4 dna ligase
    Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with <t>T4</t> DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.
    T4 Dna Ligase, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 97/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    T4 DNA Polymerase catalyzes the synthesis of DNA in the 5 →3 direction and requires the presence of template and primer This enzyme has a 3 →5 exonuclease activity which
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    Image Search Results


    Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Nucleotide sequences of integrated oligonucleotide fragments. Sequences of integrated oligonucleotide fragments with features common to all LIC-LC1 and LIC-LC2 vectors are shown. Double-stranded oligonucleotides were integrated at the restriction enzyme recognition sites indicated except for PmeI which is used to eliminate the 670-bp stuffer fragment prior to the LIC process. LIC-pPICZ-LC1/-LC2 vectors were generated by inserting AclI/SalI-restricted double-stranded oligonucleotides into BstBI/SalI-digested expression vector (cutting with AclI and BstBI creates compatible 5′ overhangs), resulting in a change of the BstBI sequence (TTCGAA to TTCGTT). The asterisk on the forward strand indicates the position of adenine (corresponding to thymine on the reverse strand) required for the generation of LIC 5′ overhangs in the presence of T4 DNA polymerase and dATP. The blue arrow indicates the TEV cleavage site suitable for the removal of the marker proteins IFP and 6xHis-tag.

    Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Generated, Expressing, Plasmid Preparation, Sequencing, Marker

    Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Journal: PLoS ONE

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    doi: 10.1371/journal.pone.0018900

    Figure Lengend Snippet: Ligation-independent cloning using LIC-IFP-compatible expression vectors. LIC vectors (LIC-LC1 and LIC-LC2) are cleaved with PmeI restriction enzyme and the released stuffer fragment (670 bp) is removed. The cleaved vector is treated with T4 DNA polymerase in the presence of dATP, whereas the PCR product (amplified open reading frame) is treated in the presence of dTTP. The asterisks indicate the position of adenine (vector) or thymine (PCR product) required for the generation of LIC-complementary 5′ overhangs. After successful annealing and transformation into E. coli , host-internal ligases and DNA polymerases close the vector and fill in the gaps, caused by the two additional nucleotides (CC, coloured in blue) upstream of the start codon (ATG), which are required to retain the reading frame. For LIC with LC1 vectors, PCR-amplified open reading frames contain a double stop codon (TAATAG); for LIC with LC2 vectors, open reading frames must not contain a stop codon to allow expression of ProteinX-TEV-IFP-6xHis fusion proteins. To provide the thymine moiety on the forward strand for dTTP/T4 DNA polymerase treatment, additional three nucleotides (GGT) are added directly at the 3′-end of the PCR-amplified open reading frame.

    Article Snippet: PCR products were treated at 22°C for 30 min with T4 DNA polymerase in the presence of dTTP, using the following reaction setup: 0.2 pmol purified PCR product, 2 µL 10× buffer 2 (NEB), 2 µL dATP (25 mM), 1 µL DTT (100 mM), 2 µL 10× BSA (10 mg/mL; NEB), 1 U T4 DNA polymerase (NEB) in a volume of 20 µL (filled up with ddH2 O).

    Techniques: Ligation, Clone Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Transformation Assay

    HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Article Snippet: The DNA was then ligated with 0.2 U of T4 DNA ligase in a final volume of 20 μl at 30°C for 30 min in the presence or absence of 5% (w/v) polyethylene glycol (PEG 8000, Sigma).

    Techniques: Plasmid Preparation, Incubation, Staining

    In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Journal: Aging and Disease

    Article Title: The Δ133p53 Isoform Reduces Wtp53-induced Stimulation of DNA Pol γ Activity in the Presence and Absence of D4T

    doi: 10.14336/AD.2016.0910

    Figure Lengend Snippet: In vitro BER assay with purified wtP53, Δ40p53 and Δ133p53 fusion proteins showing that Δ40p53 and Δ133p53 cannot induce mtBER but can attenuate mtBER activity induced by wtp53 . (A) wtP53, Δ40p53 and Δ133p53 His fusion proteins were stained with Coomassie blue (upper panel) and identified by Western blotting with anti-P53 antibodies (lower panel). (B) Purified p53, Δ40p53 and Δ133p53 protein (100, 500 and 1000 ng, lanes 3-9) or d4T (10, 50 and 300 nM, lanes 11-14) were added to BER reaction mixtures containing both whole-mitochondrial extracts obtained from H1299 cells and T4 DNA ligase. The templates were treated with T4 DNA ligase and Klenow fragment was used as a positive control (lane 15).

    Article Snippet: Klenow fragment and T4 DNA ligase were obtained from Invitrogen.

    Techniques: In Vitro, Purification, Activity Assay, Staining, Western Blot, Positive Control

    Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.

    Journal: International Journal of Molecular Medicine

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis

    doi: 10.3892/ijmm.2019.4355

    Figure Lengend Snippet: Construction of the knockdown vector pCB309-PFUFT. The FSH1 cDNA was ligated into pUC-PUT after DNA digestion by Xho I and Hin dIII to construct plasmid pUC-PFUFT. The two plasmids, pUC-PFUFT and pCB309 were digested by Spe I and Sac I and ligated with T4 DNA ligase to construct the final FSH1 double stranded RNA interference plasmid pCB309-PFUFT. FSH1, family of serine hydrolases 1.

    Article Snippet: First, the purified product of PCR for the FSH1 gene was ligated into pUC-PUT following DNA digestion with Xho I and Hin dIII, and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.).

    Techniques: Plasmid Preparation, Construct

    DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) T4 DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.

    Journal: Journal of Nucleic Acids

    Article Title: Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

    doi: 10.4061/2010/823917

    Figure Lengend Snippet: DNA end joining with immunodepleted extracts. HeLa WCE was immunodepleted for the various potential NHEJ proteins indicated, and immunodepletion (≥ 3-fold) of the target protein was confirmed by western blot (data not shown). The individual immunodepleted extracts were then assayed for DNA end-joining activity at 30°C for 2 hrs. (a) Results of DNA end-joining reactions performed in the absence of 5% PEG. (b) Results of DNA end-joining reactions performed in the presence of 5% PEG. All reactions were performed in triplicate and error bars indicate the standard deviation. (c) Wortmannin-insensitive DNA end joining is detectable in the DNA-PK cs immunodepleted HeLa WCE in the absence of PEG. Reactions were run in the absence of 5% PEG and where indicated, in the presence of 10 μ M wortmannin at 30°C for 2 hrs. (L) T4 DNA ligase positive control; (−) negative control; (ID) DNA-PK cs -immunodepleted WCE.

    Article Snippet: Materials T4 DNA ligase (10 U/μ L) was purchased from Invitrogen (Carlsbad, CA.).

    Techniques: Non-Homologous End Joining, Western Blot, Activity Assay, Standard Deviation, Positive Control, Negative Control

    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

    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

    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Journal: Nucleic Acids Research

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

    doi: 10.1093/nar/gkq572

    Figure Lengend Snippet: Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Article Snippet: PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP.

    Techniques: Ligation, Purification, Sequencing, Blocking Assay

    Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Stimulation of DNA ligation by histone H1 and deletion mutants. The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 1–15 nM ( left to right ) histone H1 (fl) or deletion mutants within the highly basic C-terminus, followed by ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: DNA Ligation, Labeling, Incubation, Ligation, Electrophoresis

    Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: Histone H1 inhibits the ability of HMGB1 to bend DNA. A , formation of DNA circles by HMGB1 is inhibited by the full-length histone H1 (DNA circularization assay). The 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 5 nM HMGB1, followed by titration with increasing concentrations of H1 (0.2–15 nM, left to right ) and ligation by T4 DNA ligase. Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. Panels B - E , DNA circularization assays in the presence of the full-length histone H1(fl) or peptides H1Δ24, H1Δ48 and H1Δ72. The percentage of DNA circles by reduced or oxidized HMGB1 or HMGB1ΔC (50 nM) in the presence of increasing concentrations of H1 or H1 peptides (1–15 nM, left to right ) is indicated. The percentage of the minicircles formed by HMGB1 or HMGB1ΔC in the absence of H1 or peptides was arbitrary set to 100%. Oxidized HMGB1 or HMGB1ΔC proteins are indicated in red.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Labeling, Incubation, Titration, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1ΔC on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was pre-incubated with 2, 5, 10, 15, 25, 50 and 100 nM of HMGB1 lacking the acidic C-tail (HMGB1ΔC, left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced (black triangle) or oxidized (empty triangle) HMGB1ΔC, as compared to DNA circles formed under the same conditions by reduced (black circles) or oxidized (empty circles) full-length HMGB1. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1ΔC, oxidized HMGB1ΔC, and HMGB1ΔC(F37A). Concentrations of proteins were 5, 10, 25, 50 and 100 nM ( left to right ).

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Incubation, Ligation, Electrophoresis

    The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Journal: PLoS ONE

    Article Title: Histone H1 Differentially Inhibits DNA Bending by Reduced and Oxidized HMGB1 Protein

    doi: 10.1371/journal.pone.0138774

    Figure Lengend Snippet: The effect of oxidization and mutation of Cys22/Cys44 or Phe37 of HMGB1 on DNA bending. A , the 5´-end 32 P-labeled 123-bp DNA fragment (~1 nM) was preincubated with 2, 5, 10, 15, 25, 50 and 100 nM HMGB1 proteins ( left to right ), followed by ligation by T4 DNA ligase (DNA circularization assay). Deproteinised DNA samples were separated by electrophoresis on 5% non-denaturing polyacrylamide gels in 0.5x TBE buffer. B , percentage of DNA circles formed by reduced HMGB1, oxidized HMGB1 or HMGB1(Cys22A/Cys44A) mutant. The percentage of the minicircles formed at 100 nM HMGB1 was arbitrary set to 100% (each of the curves represent an average of three independent experiments). C , representative circularization assay using reduced HMGB1 and HMGB1(F37A) mutant (5, 20, 50 and 100 nM HMGB1, left to right ). C22/C44, HMGB1(Cys22A/Cys44A) mutant.

    Article Snippet: In agreement with previous reports [ , ], histone H1 could stimulate formation of linear multimers by T4 DNA ligase at low H1-to-DNA ratios.

    Techniques: Mutagenesis, Labeling, Ligation, Electrophoresis

    Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

    doi: 10.1016/j.bbrep.2016.10.006

    Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles in the absence of a magnetic field on the ambient temperature. The ordinate axis represents the ligation efficiency, which is normalized by that at 16 °C. The standard deviations are obtained from 6 independent experiments.

    Article Snippet: 2.1 Immobilization of T4 DNA ligase on ferromagnetic particles We immobilized T4 DNA ligase (EC 6.5.1.1, Takara Bio Inc.) on ferromagnetic iron particles, the surface of which had not been modified with any molecules (Spherical Ferromagnetic Iron Powder, Catalog No. 19844-1, Polysciences Inc.).

    Techniques: DNA Ligation, Ligation

    Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

    Journal: Biochemistry and Biophysics Reports

    Article Title: Efficient DNA ligation by selective heating of DNA ligase with a radio frequency alternating magnetic field

    doi: 10.1016/j.bbrep.2016.10.006

    Figure Lengend Snippet: Dependence of the efficiency of DNA ligation using T4 DNA ligase immobilized on ferromagnetic particles under an ac magnetic field of 0.34 MHz on the amplitude of the magnetic field. The ambient temperature is 16 °C. The ordinate axis represents the ligation efficiency under an ac magnetic field, which is normalized by that in the absence of a magnetic field. The inset shows the ligation efficiency under the ac magnetic field as a function of the average surface temperature of ferromagnetic particles, noting that the surface temperature increases with an increase in the field amplitude. The standard deviations are obtained from 6 independent experiments.

    Article Snippet: 2.1 Immobilization of T4 DNA ligase on ferromagnetic particles We immobilized T4 DNA ligase (EC 6.5.1.1, Takara Bio Inc.) on ferromagnetic iron particles, the surface of which had not been modified with any molecules (Spherical Ferromagnetic Iron Powder, Catalog No. 19844-1, Polysciences Inc.).

    Techniques: DNA Ligation, Ligation

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Journal: Nucleic Acids Research

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    doi: 10.1093/nar/gkl449

    Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR.

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    LIC procedure using pMCSG vectors. All MCSG vectors contain an Ssp I site (AATATT) positioned immediately after the sequence encoding the TEV protease recognition site. Cleavage with Ssp I (a blunt cutter) followed by treatment with T4 DNA polymerase in

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins 1

    doi: 10.1007/978-1-59745-196-3_7

    Figure Lengend Snippet: LIC procedure using pMCSG vectors. All MCSG vectors contain an Ssp I site (AATATT) positioned immediately after the sequence encoding the TEV protease recognition site. Cleavage with Ssp I (a blunt cutter) followed by treatment with T4 DNA polymerase in

    Article Snippet: dCTP (100 mM) (Promega cat. no. U1221) Dithiothreitol (DTT, 100 mM), molecular biology grade (Sigma cat. no. D-9779) T4 DNA polymerase, LIC-qualified (Novagen cat. no. 70099) 10× T4 polymerase buffer (included with polymerase)

    Techniques: Sequencing

    Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.

    Journal: Nucleic Acids Research

    Article Title: Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis

    doi: 10.1093/nar/gkz015

    Figure Lengend Snippet: Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.

    Article Snippet: The same DNA products were obtained by nicking the DNA with Nt-Bst NBI endonuclease (NEB) and sealing the nicks afterward with phage T4 DNA ligase (NEB) in presence of 250 μg/ml of chloroquine.

    Techniques: Electrophoresis, Western Blot, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis

    (a) AFM scan and SEM image (insert) of the UV curable resin stamp possessing a positive tone of the 2-D nanochannels. Channels were imprinted into PMMA with dimensions (width × depth) of nc 1 = 300 × 200 nm, nc 2 = 250 × 155 nm, nc 3 = 190 × 95 nm, nc 4 = 150 × 60 nm and nc 5 = 110 × 25 nm. (b) Bar graphs showing the signal-to-noise ratio (SNR) at 2 s exposure time for the devices with untreated PMMA substrate enclosed with a plasma treated COC cover plate, U-PMMA/(PL-COC), and plasma treated substrate enclosed with a plasma treated PMMA cover plate, PL-PMMA/(PL-PMMA) filled with a 5 mM FITC solution. The error bars represent the standard deviation in measurements from ten separate devices. Insert shows unprocessed images of the seeding test for U-PMMA/(PL-COC). (c) Unprocessed representative frames of T4 DNA molecules elongated in enclosed nanochannels for the hybrid devices. Images were acquired at 10 ms exposure time with the driving field turned-off. Note that nc 6 = 35 × 35 nm. (d) Log-log plot showing the T4 DNA extension as a function of the geometric average depth of the nanochannels. The DNA extension was normalized to a total contour length (L c ) of 64 µm for the dye-labeled molecules. The red and blue dashed lines are the deGennes and Odijk predictions, respectively. The black solid line is the best power-law fit to the data points obtained from the nanochannels with an average geometric depth range of 53 nm to 200 nm.

    Journal: Lab on a chip

    Article Title: High Process Yield Rates of Thermoplastic Nanofluidic Devices using a Hybrid Thermal Assembly Technique

    doi: 10.1039/c4lc01254b

    Figure Lengend Snippet: (a) AFM scan and SEM image (insert) of the UV curable resin stamp possessing a positive tone of the 2-D nanochannels. Channels were imprinted into PMMA with dimensions (width × depth) of nc 1 = 300 × 200 nm, nc 2 = 250 × 155 nm, nc 3 = 190 × 95 nm, nc 4 = 150 × 60 nm and nc 5 = 110 × 25 nm. (b) Bar graphs showing the signal-to-noise ratio (SNR) at 2 s exposure time for the devices with untreated PMMA substrate enclosed with a plasma treated COC cover plate, U-PMMA/(PL-COC), and plasma treated substrate enclosed with a plasma treated PMMA cover plate, PL-PMMA/(PL-PMMA) filled with a 5 mM FITC solution. The error bars represent the standard deviation in measurements from ten separate devices. Insert shows unprocessed images of the seeding test for U-PMMA/(PL-COC). (c) Unprocessed representative frames of T4 DNA molecules elongated in enclosed nanochannels for the hybrid devices. Images were acquired at 10 ms exposure time with the driving field turned-off. Note that nc 6 = 35 × 35 nm. (d) Log-log plot showing the T4 DNA extension as a function of the geometric average depth of the nanochannels. The DNA extension was normalized to a total contour length (L c ) of 64 µm for the dye-labeled molecules. The red and blue dashed lines are the deGennes and Odijk predictions, respectively. The black solid line is the best power-law fit to the data points obtained from the nanochannels with an average geometric depth range of 53 nm to 200 nm.

    Article Snippet: All images were analyzed using Fiji software. λ-DNA (Promega Corporation) and T4 DNA (Wako Chemicals) were stained with the bis-intercalating dye, YOYO-1 (Molecular Probes, Eugene, OR) at a base-pair/dye ratio of 5:1 in a buffer solution of 1× TBE (89 mM Tris, 89 mM Borate, 1 mM EDTA) with the addition of 4% v/v β-mercaptoethanol used as a radical scavenger to minimize photo-induced damage (photobleaching and/or photonicking).

    Techniques: Standard Deviation, Mass Spectrometry, Labeling

    15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 15% denaturing PAGE for the ligation products of linkers A–B, C–D and linkers G–H. PAGE (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5x TBE) was run in 0.5 x TBE, 25°C, 100 V for 3.5 hrs in ( A )–( F ), or 4.3 hrs in ( G ). The ligation products were indicated by the arrows. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas). Lane M1: DNA marker I plus oligo 15. ( A ) The ligation products joined by using T4 DNA ligase from Fermentas. Lane 1: the ligation products of linkers C–D preincubated with T4 DNA ligase; Lane 2: the ligation products of linkers C–D without the preincubation; Lane 4: the ligation products of linkers A–B; Lanes 3 and 5: the negative controls. ( B ) The ligation products joined by using T4 DNA ligase from Takara. Lanes 1–3∶0.5, 1, and 2 µl of 1 µM oligo 15, respectively; Lanes 4 and 6: the ligation products of linkers A–B; Lane 8: the ligation products of linkers C–D. Lanes 5, 7, and 9: the negative controls. ( C ) The ligation products joined by using T4 DNA ligase from Promega. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products joined by using E. coli DNA ligase from Takara. Lanes 1 and 3: the ligation products of linkers A–B, and C–D, respectively; Lanes 2 and 4: the negative controls. ( E ) The ligation products of linkers A–B joined in T4 DNA ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lanes 1–3: the ligase reaction mixture with 7.5 mM (NH 4 ) 2 SO 4 , 3.75 mM (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively; Lane 4: the negative control. ( F ) The ligation products of the phosphorylated linkers A–B and C–D joined by using T4 and E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of the phosphorylated linkers A–B joined by using T4 and E. coli DNA ligase, respectively; Lanes 3 and 5: the ligation products of the phosphorylated linkers C–D joined by using T4 and E. coli DNA ligase, respectively; Lanes 6 and 7: the ligation products of linkers A–B and C–D, respectively; Lanes 8 and 9: the negative controls of lanes 6 and 7, respectively. ( G ) The ligation products of linkers A–B and the phosphorylated linkers G–H. Lanes 1 and 2: the ligation products of linkers A–B and the ligation products of the phosphorylated linkers G–H plus the negative control of linkers A–B, respectively; Lane 3: the negative control of linkers G–H plus the negative control of linkers A–B. The band from the ligation products of the phosphorylated linkers G–H run a little more slowly than that of linkers A–B. The sequences of linkers G and H are similar to those of linkers A and B, respectively. But there is a 1-base deletion at the 5′ end of each of linkers G and H.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B treated with CIAP. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15. The ligases used in ( A )–( C ) were T4 DNA ligases. The ligases used in ( D )–( E ) were E. coli DNA ligases. ( A ) CIAP was inactivated at 75°C for 15 min. Lanes 1 and 5∶1 µl of 1 µM oligo 15; Lanes 2: CIAP was inactivated at 75°C for 15 min; Lane 3: the positive control without CIAP treatment; Lane 4: the negative control without ligase. ( B ) CIAP was inactivated at 85°C for 25 min and 45 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 25 min and 45 min, respectively; Lane 5: the negative control without ligase. ( C ) CIAP was inactivated at 85°C for 65 min and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 min and 90 min, respectively; Lane 5: the negative control without ligase. ( D ) CIAP was inactivated at 85°C for 45 min. Lanes 1 and 3: the positive control without CIAP treatment and the negative control without ligase, respectively; Lane 2: CIAP was inactivated at 85°C for 45 min. ( E ) CIAP was inactivated at 85°C for 65 and 90 min. Lanes 1 and 3: the positive controls without CIAP treatment; Lanes 2 and 4: CIAP was inactivated at 85°C for 65 and 90 min, respectively; Lane 5: the negative control without ligase.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Positive Control, Negative Control

    12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: 12% denaturing PAGE for the ligation products of linkers A–B, C–D, and E–F. PAGE (10×10×0.03 cm, A:B = 19∶1, 7 M urea and 0.5 x TBE) was run in 0.5 x TBE, 25°C, 200 V for 1.7 hrs for the ligation products of linkers A–B and C–D, or 100 V for 3.5 hrs for those of linkers E–F. The arrows indicate the ligation products. Lane M: DNA marker I (GeneRuler™ 50 bp DNA ladder, Fermentas); Lane M1: DNA marker I +1 µl of 1 µM oligo 15; Lane M2: pUC19 DNA/MspI Marker (Fermentas). ( A ) The ligation products joined by using T4 DNA ligase from Takara and Fermentas. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 6: the ligation products of linkers A–B joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 5 bands. Of them, bands 1 and 2 were from oligos 4 and 1, respectively. Band 3 was from both oligos 2 and 3. Band 4 was unknown. Perhaps it might be the intermixtures of oligos 1–4. Band 5 was the denatured ligation products of linkers A–B; Lanes 4 and 8: the ligation products of linkers C–D joined by using T4 DNA ligase from Takara and Fermentas, respectively. We could see 4 bands. Of them, bands 6 and 7 were from both oligos 6 and 7, and both oligos 5 and 8, respectively. Band 8 was the denatured ligation products of linkers C–D. Band 9 was unknown. Perhaps it might be the intermixtures of oligos 5–8 and the double-strand ligation products of linkers C–D; Lanes 3, 5, 7, and 9: the negative controls. ( B ) The ligation products of linkers A–B and C–D joined by using T4 DNA ligase from Promega and the ligation products of linkers A–B joined in the ligase reaction mixture containing (NH 4 ) 2 SO 4 . Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the denatured ligation products of linkers A–B, and C–D, respectively. T4 DNA ligase was from Promega; Lanes 6 and 7: the ligation products of linkers A–B joined in the ligase reaction mixture without (NH 4 ) 2 SO 4 and with (NH 4 ) 2 SO 4 , respectively. T4 DNA ligase used was from Takara; Lanes 3, 5, and 8: the negative controls. ( C ) The ligation products of linkers A–B and C–D joined by using E. coli DNA ligase. Lane 1∶1 µl of 1 µM oligo 15; Lanes 2 and 4: the ligation products of linkers A–B, and C–D, respectively; Lanes 3 and 5: the negative controls. ( D ) The ligation products of linkers E–F joined in the ligase reaction mixture with (NH 4 ) 2 SO 4 . The ligase was T4 DNA ligase (Fermentas). Lane 1: pUC19 DNA/MspI Marker plus 2 µl of ligation products of linkers E–F; Lanes 2 and 3: the ligation products of linkers E–F joined in the ligase reaction mixtures with (NH 4 ) 2 SO 4 , and without (NH 4 ) 2 SO 4 , respectively. We could see 3 bands. Bands 10 and 11 are from both oligos 9 and 12, and both oligos 10 and 11, respectively; Band 12 is the ligation products of linkers E–F; Lane 4: the negative control. ( E ) The ligation products of linkers E–F joined by using E. coli DNA ligase. Lane 1: the ligation products of linkers E–F. Lane 2: the negative control. ( F ) The ligation products of linkers A–B preincubated with T4 PNK in the E. coli DNA ligase reaction mixture without ATP. The ligase was E. coli DNA ligase (Takara). Lane 1∶1 µl of 1 µM oligo 15; Lane 2: linkers A–B were not preincubated with T4 PNK; Lane 3: linkers A–B were preincubated with T4 PNK; Lane 4: the negative control.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Ligation, Marker, Negative Control

    The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Journal: PLoS ONE

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain

    doi: 10.1371/journal.pone.0039251

    Figure Lengend Snippet: The radioautograph of oligo 11 phosphorylated by T4 DNA ligase. The oligo 11 was phosphorylated by using commercial T4 DNA ligase. The phosphorylation products were loaded on a 15% denaturing PAGE gel (10×10×0.03 cm, A:B = 29∶1, 7 M urea, 0.5 x TBE). Electrophoresis was run in 0.5 x TBE at 100 V and 25°C for 3 hrs. The gel was dried between two semipermeable cellulose acetate membranes and radioautographed at −20°C for 1–3 days. The arrows indicate the phosphorylation products. The positive controls were oligo 11 phosphorylated by T4 PNK. ( A ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lanes 2 and 4: the negative controls without ligase, and without oligo 11, respectively; Lane 3: the phosphorylation products of oligo 11 by T4 DNA ligase. ( B ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 15 min, 30 min, and 60 min, respectively. Lanes 9 and 10: the negative controls without ligase, and without oligo 11, respectively. ( C ) Oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. Lanes 1 and 5: the positive controls; Lane 2: the phosphorylation products of oligo 11 by T4 DNA ligase; Lanes 3 and 4: the negative controls without ligase, and without oligo 11, respectively; Lanes 6, 7, and 8: oligo 11 treated with CIAP was phosphorylated by T4 DNA ligase. CIAP was inactivated at 85°C for 60 min, 15 min, and 30 min, respectively. ( D ) Oligos 11 and 12 were phosphorylated by T4 DNA ligase at 37°C for 1 hr. Lane 1: oligos 11 and 12 were phosphorylated by T4 PNK; Lane 2: oligos 11 and 12 were phosphorylated by T4 DNA ligase; Lane 3: oligo 11 were phosphorylated by T4 DNA ligase; Lane 4: the negative control without ligase. ( E ) Oligo 11 was phosphorylated by T4 DNA ligase at 37°C for 2 hrs. 1 x TE and 10% SDS were not added to the phosphorylation products before phenol/chloroform extraction. Lane 1: the positive control; Lanes 2 and 3: the phosphorylation products of oligo 11 by T4 DNA ligase and the negative controls without ligase, respectively.

    Article Snippet: Ligations of the linkers with 5′-OH ends The ligations of linkers A–B, C–D, and E–F by using T4 DNA ligase were performed in 100 µl of T4 DNA ligase reaction mixture containing 1 x T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 0.5 mM ATP; pH 7.8 at 25°C), 1 µM of each oligo, and 0.25 Weiss units/µl of T4 DNA ligase (Fermentas, Lithuania; Promega, USA; and Takara, Japan).

    Techniques: Polyacrylamide Gel Electrophoresis, Electrophoresis, Negative Control, Positive Control

    Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    Journal: PLoS ONE

    Article Title: A Rapid Cloning Method Employing Orthogonal End Protection

    doi: 10.1371/journal.pone.0037617

    Figure Lengend Snippet: Efficient synthon assembly with split-and-pool reactions. (A) Equimolar amounts of BsaI or BsmBI deprotected 13 FNIII synthons were incubated with 1 unit of T4 ligase and product formation was assessed at different time points (left panel) or after 15 min in buffer conditions with and without 15% (w/v) PEG6000 (right panel). (B) No significant differences in assembly efficiency are observed after 15′ incubation at ligase concentrations ranging from 1 to 10 units. (C) Performance of split-and-pool assembly in comparison to sequential approaches. Within one day the comprehensive series of ( 13 FNIII) 1 to ( 13 FNIII) 8 repeats can be assembled with the split-and-pool approach (spectrum circles) and ligated into the pShuttle vector. After a single cloning step expression plasmid is obtained on day 3. In comparison, sequential assembly with e.g. the BamHI/BglII system requires 12 days to obtain the ( 13 FNIII) 8 construct.

    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.

    Techniques: Incubation, Plasmid Preparation, Clone Assay, Expressing, Construct

    T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Deficient DNA-ligase activity in the metabolic disease tyrosinemia type I

    doi:

    Figure Lengend Snippet: T4 DNA-ligase activity in the presence of SA. T4 DNA-ligase was treated or not with increasing SA concentrations (15 min at 25°C) and then incubated at 37°C for 1 min with the oligo substrate. ( A ) The oligo(dT) 16 multimers were separated in polyacrylamide/urea gels: T4 DNA-ligase without SA (lane 1) or incubated with increasing SA concentrations of 2.5(2), 5(3), 10(4), and 20(5) μM. ( B ) The activity was quantitated using an InstantImager (Packard). ( C ) Inhibition of enzyme-adenylate formation by SA. T4 DNA-ligase was incubated or not with increasing SA concentrations (15 min at 25°C) before the addition of [α- 32 P]ATP. The enzyme adenylate complexes were separated by electrophoresis and detected by autoradiography.

    Article Snippet: T4 DNA-ligase was from Boehringer Mannheim.

    Techniques: Activity Assay, Incubation, Inhibition, Electrophoresis, Autoradiography

    Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Journal: Journal of Virology

    Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

    doi: 10.1128/JVI.75.22.10573-10581.2001

    Figure Lengend Snippet: Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Article Snippet: Reactions were carried out at 37°C for 30 min in a final volume of 20 μl containing 400 ng of TNAs, 100 μM each dNTP, and 2 U of T4 DNA polymerase (Boehringer Mannheim) in the incubation buffer supplied, then the concentration of each dNTP was brought to 200 μM, and incubation was prolonged for another 30 min.

    Techniques: Incubation, Transgenic Assay, Infection, Migration

    3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    doi: 10.1093/dnares/dsy037

    Figure Lengend Snippet: 3′ Branch ligation by T4 DNA ligase at non-conventional DNA ends formed by nicks, gaps, and overhangs. (a) Schematic representation of ligation assay with different DNA accepter types. The blunt-end DNA donor (blue) is a synthetic, partially dsDNA molecule with dideoxy 3 ′ -termini (filled circles) to prevent DNA donor self-ligation. The long arm of the donor is 5 ′- phosporylated. The DNA acceptors were assembled using 2 or 3 oligos (black, red, and orange lines) to form a nick (without phosphates), a gap (1 or 8 nt), or a 36-nt 3 ′ -recessive end. All strands of the substrates are unphosphorylated, and the scaffold strand is 3 ′ dideoxy protected. (b) Analysis of the size shift of ligated products of substrates 1, 2, 3, and 4, respectively, using a 6% denaturing polyacrylamide gel. Reactions were performed according to the optimized condition. The negative no-ligase controls (lanes 1, 3, 4, 6, 7, 9, 10, 12, and 13) were loaded at 1 or 0.5× volume of corresponding experimental assays. If ligation occurs, the substrate size is shifted up by 22 nt. Red arrowheads correspond to the substrate, and purple arrowheads correspond to donor-ligated substrates. Donor and substrate sequences in Supplementary Table S1 . (c) Expected sizes of substrate and ligation product and approximate ligation efficiency in each experimental group. The intensity of each band was estimated using ImageJ and normalized by its expected size. Ligation efficiency was estimated by dividing the normalized intensity of ligated products by the normalized total intensity of ligated and unligated products.

    Article Snippet: T4 DNA ligase was also reported to ligate specific or degenerate single-stranded oligos to partially single-stranded substrates through hybridization., Here, we demonstrated a non-conventional T4 DNA ligase-mediated ligation that does not require complimentary base pairing and can ligate a blunt-end DNA donor to the 3 ′ OH end of a duplex DNA acceptor at 3 ′ -recessed strands, gaps, or nicks ( a).

    Techniques: Ligation

    3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: 3′ Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3′OH ends in DNA or RNA

    doi: 10.1093/dnares/dsy037

    Figure Lengend Snippet: 3′ Branch ligation at the 3′ end of RNA in DNA/RNA hybrid. Schematic representation of 3′-branch ligation on a DNA/RNA hybrid with a 20‐bp complimentary region. We tested whether blunt-end DNA donors would ligate to the 3 ′ -recessive end of DNA and/or to the 3 ′ -recessive end of RNA. DNA(ON-21) hybridizes with the RNA strand (a), whereas DNA(ON-23) cannot hybridize with the RNA strand (b). (c, d) Gel analysis of size shift of ligated products using 6% denaturing polyacrylamide gel. The red arrowheads correspond to the RNA substrate (29 nt), and the green arrowhead corresponds to DNA substrate (80 nt). The purple arrowhead corresponds to donor-ligated RNA substrates. If ligation occurs, the substrate size would shift up by 20 nt. (c) Lanes 1 and 2, experimental duplicates; lanes 7–10, no-ligase controls; 10% PEG was added with T4 DNA ligase. (d) Lane 1, no-ligase control; lanes 2, 3, and 8, T4 DNA ligase with 10% PEG; lanes 4, 5, and 9, T4 RNA ligase 1 with 20% DMSO; lanes 6, 7, and 10, T4 RNA ligase 2 with 20% DMSO.

    Article Snippet: T4 DNA ligase was also reported to ligate specific or degenerate single-stranded oligos to partially single-stranded substrates through hybridization., Here, we demonstrated a non-conventional T4 DNA ligase-mediated ligation that does not require complimentary base pairing and can ligate a blunt-end DNA donor to the 3 ′ OH end of a duplex DNA acceptor at 3 ′ -recessed strands, gaps, or nicks ( a).

    Techniques: Ligation

    The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Journal: PLoS ONE

    Article Title: GreenGate - A Novel, Versatile, and Efficient Cloning System for Plant Transgenesis

    doi: 10.1371/journal.pone.0083043

    Figure Lengend Snippet: The Golden Gate principle. A) Type IIS restriction endonucleases, such as Bsa I, have a distinct, non-palindromic recognition site (red) and asymmetrically cut at a precisely defined distance regardless of the local sequence (green). Bsa I for instance creates a four base 5′-overhang starting from the second nucleotide downstream of the recognition site. B) A Golden Gate style cloning system requires two types of components, a destination vector and entry vectors containing the modules to be assembled. Each vector carries two recognition sites for the type IIS endonuclease (red) flanking the counter-selective marker on the destination vector and the modules on the entry vectors, respectively. Destination and entry vectors confer different markers for bacterial selection. The sequences in purple, blue and green represent the cutting sites. C) The orientation and position of the recognition sites is such that after digestion they remain with the backbone of the entry vectors, but are excised from the destination vector along with the counter-selectable marker ( ccdB ). D) The single stranded overhangs generated by the endonuclease can anneal to complementary sequences and be covalently linked by T4 DNA ligase. During the Golden Gate reaction in the presence of endonuclease and ligase the desired final product, but also the original vectors or a plethora of side-products (one of them shown at the bottom) can be created. However, only the desired final product is resistant to further endonucleolytic cleavage, whereas all other molecules will be cut again and again and thus will disappear from the reaction over time.

    Article Snippet: PCR products and intermediate plasmids were cut by conventional restriction endonucleases (obtained from Fisher Scientific - Germany GmbH, Schwerte, Germany) and ligated with T4 DNA ligase (Fisher Scientific - Germany GmbH, Schwerte, Germany).

    Techniques: Sequencing, Clone Assay, Plasmid Preparation, Marker, Selection, Generated

    Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or T4 DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.

    Journal: Nucleic Acids Research

    Article Title: Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

    doi: 10.1093/nar/gkg892

    Figure Lengend Snippet: Effect of DNA ligase addition on the DNA ligase-[α- 32 P]AMP reduction. ( A ) Cell-free DNA repair assay was carried out for 60 min with HeLa S3 cell extracts containing DNA ligase-[α- 32 P]AMP. Plasmid DNA containing either γ-ray-induced SSIs (γ-SSI) or alkylated base damage induced by MNNG was used for the assay. The reactions also contained Lig I, Lig III or T4 DNA ligase (T4 Lig). After the repair reaction, the mixtures were fractionated on an SDS–7.5% polyacrylamide gel, and 32 P activity was visualized by autoradiography. Alternatively, 32 P activities were measured with an AlphaImager (Packard) and quantified results are shown in ( B ) (γ-SSI) and ( C ) (MNNG). The results shown are from one of four independent experiments. The amount of Lig I-[ 32 P]AMP (non-damaged DNA) was calculated as 100%.

    Article Snippet: The activity of the resulting DNA ligases was determined using T4 DNA ligase (Amersham Biosciences) as a standard and expressed in Weiss units.

    Techniques: Plasmid Preparation, Activity Assay, Autoradiography

    Assay design and preparation of cell-free extracts containing DNA ligase-[α- 32 P]AMP. ( A ) Whole cell-free extracts were treated with PPi to remove AMP from DNA ligases. PPi was then dialyzed out and the dialyzed extracts were incubated with [α- 32 P]ATP to form DNA ligase- [α- 32 P]AMP. Extracts containing DNA ligase-[α- 32 P]AMP were used for cell-free DNA repair assays. When the DNA ligase-[α- 32 P]AMP is used to rejoin DNA nicks, [α- 32 P]AMP is released from the DNA ligase. ( B ) Cell-free extracts containing DNA ligase-[α- 32 P]AMP, recombinant Lig I- [α- 32 P]AMP, Lig III-[α- 32 P]AMP and T4 DNA ligase (T4 Lig)-[α- 32 P]AMP were fractionated by SDS–7.5% PAGE and 32 P activity was visualized by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

    doi: 10.1093/nar/gkg892

    Figure Lengend Snippet: Assay design and preparation of cell-free extracts containing DNA ligase-[α- 32 P]AMP. ( A ) Whole cell-free extracts were treated with PPi to remove AMP from DNA ligases. PPi was then dialyzed out and the dialyzed extracts were incubated with [α- 32 P]ATP to form DNA ligase- [α- 32 P]AMP. Extracts containing DNA ligase-[α- 32 P]AMP were used for cell-free DNA repair assays. When the DNA ligase-[α- 32 P]AMP is used to rejoin DNA nicks, [α- 32 P]AMP is released from the DNA ligase. ( B ) Cell-free extracts containing DNA ligase-[α- 32 P]AMP, recombinant Lig I- [α- 32 P]AMP, Lig III-[α- 32 P]AMP and T4 DNA ligase (T4 Lig)-[α- 32 P]AMP were fractionated by SDS–7.5% PAGE and 32 P activity was visualized by autoradiography.

    Article Snippet: The activity of the resulting DNA ligases was determined using T4 DNA ligase (Amersham Biosciences) as a standard and expressed in Weiss units.

    Techniques: Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Activity Assay, Autoradiography

    Optimization of D-probe complementary sequence length. (A) Complementary sequences of four probe sets for miR-143. (B) The fluorescence intensities of D-probes bound to C-probes in the presence of T4 DNA ligase. Data represent the mean ± S.E. (n = 3). (C) Correlation between input miR-143 and the signal of D-probe-143-(8) in the presence (filled circle) or absence (open circle) of T4 DNA ligase. The upper axis indicates the final concentration of input miR-143 in the hybridization chamber while the lower axis indicates the total amount of input miR-143. Data in the linear range were fitted by a linear expression (gray lines). Data represent the mean ± S.E. (n = 3).

    Journal: PLoS ONE

    Article Title: Label-Free Quantification of MicroRNAs Using Ligase-Assisted Sandwich Hybridization on a DNA Microarray

    doi: 10.1371/journal.pone.0090920

    Figure Lengend Snippet: Optimization of D-probe complementary sequence length. (A) Complementary sequences of four probe sets for miR-143. (B) The fluorescence intensities of D-probes bound to C-probes in the presence of T4 DNA ligase. Data represent the mean ± S.E. (n = 3). (C) Correlation between input miR-143 and the signal of D-probe-143-(8) in the presence (filled circle) or absence (open circle) of T4 DNA ligase. The upper axis indicates the final concentration of input miR-143 in the hybridization chamber while the lower axis indicates the total amount of input miR-143. Data in the linear range were fitted by a linear expression (gray lines). Data represent the mean ± S.E. (n = 3).

    Article Snippet: Silane-PEG-Maleimide (MW 5000), betaine solution, PEG 6000 solution, bovine serum albumin (BSA) and T4 DNA ligase were purchased from NANOCS Inc. (Boston, MA, USA), WAKO (Osaka, Japan), Hampton Research (Aliso Viejo, CA, USA), Sigma Aldrich Japan (Tokyo, Japan) and TAKARA (Shiga, Japan), respectively.

    Techniques: Sequencing, Fluorescence, Concentration Assay, Hybridization, Expressing

    Experimental design of the LASH assay. (A) Schematic representation of the ligase-assisted sandwich hybridization assay (LASH assay) for detection of non-labeled miRNA. A target miRNA hybridizes to complimentary D-probe and C-probe, resulting in a tertiary complex. Both C-probe and D-probe are capped with a phosphate group at the 5′ end and a hydroxyl group at the 3′ end; this permits T4 DNA ligase to ligate the 5′ end of C-probe to the 3′ end of D-probe, and to ligate the 5′ end of D-probe to the 3′ end of the target miRNA. (B) A bright-field image of droplets spotted on a coverslip by an ink-jet machine. Scale bar, 400 µm. (C) Fluorescence images of 6-FAM-labeled C-probe (left) immobilized on a substrate and Alexa647-labeled D-probe (right) bound to C-probe in the presence of 1.25 nM miR-143. Scale bar, 100 µm.

    Journal: PLoS ONE

    Article Title: Label-Free Quantification of MicroRNAs Using Ligase-Assisted Sandwich Hybridization on a DNA Microarray

    doi: 10.1371/journal.pone.0090920

    Figure Lengend Snippet: Experimental design of the LASH assay. (A) Schematic representation of the ligase-assisted sandwich hybridization assay (LASH assay) for detection of non-labeled miRNA. A target miRNA hybridizes to complimentary D-probe and C-probe, resulting in a tertiary complex. Both C-probe and D-probe are capped with a phosphate group at the 5′ end and a hydroxyl group at the 3′ end; this permits T4 DNA ligase to ligate the 5′ end of C-probe to the 3′ end of D-probe, and to ligate the 5′ end of D-probe to the 3′ end of the target miRNA. (B) A bright-field image of droplets spotted on a coverslip by an ink-jet machine. Scale bar, 400 µm. (C) Fluorescence images of 6-FAM-labeled C-probe (left) immobilized on a substrate and Alexa647-labeled D-probe (right) bound to C-probe in the presence of 1.25 nM miR-143. Scale bar, 100 µm.

    Article Snippet: Silane-PEG-Maleimide (MW 5000), betaine solution, PEG 6000 solution, bovine serum albumin (BSA) and T4 DNA ligase were purchased from NANOCS Inc. (Boston, MA, USA), WAKO (Osaka, Japan), Hampton Research (Aliso Viejo, CA, USA), Sigma Aldrich Japan (Tokyo, Japan) and TAKARA (Shiga, Japan), respectively.

    Techniques: Hybridization, Labeling, Fluorescence

    Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Journal: Nucleic Acids Research

    Article Title: The role of DNA dependent protein kinase in synapsis of DNA ends

    doi: 10.1093/nar/gkg889

    Figure Lengend Snippet: Ligation in the presence of DNA-PK requires ATP hydrolysis and an active DNA-PK CS kinase. ( A ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1–3) or the presence (lanes 4 and 5) of DNA-PK. ATP or AMP-PNP was present as indicated. Ligation products were separated by agarose gel electrophoresis. The nature of the ligation products, identified as intra- or inter-molecular ligation products, was confirmed by exonuclease V digestion. Note that intra-molecular ligation products can be either ligated on one strand (open circular form) or on both strands (covalently closed circular form). ( B ) An overall labeled DNA substrate with cohesive ends was incubated with E.coli DNA ligase, either in the absence (lanes 1–4) or the presence (lanes 5 and 6) of DNA-PK. ATP and/or NAD + were present as indicated. ( C ) An overall labeled DNA substrate with cohesive ends was incubated with T4 DNA ligase, either in the absence (lanes 1 and 2) or the presence (lanes 3 and 4) of DNA-PK. All reaction mixtures contained ATP. The DNA-PK CS kinase inhibitor wortmannin was added in lane 4. Total levels of ligation products in all lanes were decreased in comparison with (A), due to the presence of DMSO in the reaction mixtures. ( D ) Wortmannin inhibits autophosphorylation of DNA-PK CS . Incorporation of radiolabeled phosphate into DNA-PK CS was determined in the absence and presence of 1 or 10 µM wortmannin. Even 1 µM wortmannin completely inhibits DNA-PK CS autophosphorylation.

    Article Snippet: First, we used AMP-PNP, an ATP analog that supports activity of T4 DNA ligase, but cannot function as a cofactor for DNA-PKCS (Fig. A).

    Techniques: Ligation, Labeling, Incubation, Agarose Gel Electrophoresis

    Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with T4 DNA polymerase, which robustly

    Journal: The Journal of Biological Chemistry

    Article Title: The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex *

    doi: 10.1074/jbc.M114.570333

    Figure Lengend Snippet: Same efficiency of the extension of DNA and RNA primers on hetero-homopolymeric hybrid and heteropolymeric DNA templates by the p180ΔN-core. For control of the full extension of the primers, we used reactions with T4 DNA polymerase, which robustly

    Article Snippet: T4 DNA polymerase (Promega Corporation; stock concentration 25 n m ) was used as a control for the primer extensions on hetero-DNA and RNA.

    Techniques:

    A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using T4 DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).

    Journal: The American Journal of Pathology

    Article Title: Early Necrotic DNA Degradation

    doi:

    Figure Lengend Snippet: A–C : Comparison of apoptotic ( A , B ) and necrotic ( C ) thymus stained by DAPI (blue fluorescence) and by in situ ligation using oligonucleotide probes with single dA overhangs (red fluorescence). A and C are dual-stained images, B is a single-stained (DAPI) image and is provided for the easy comparison of nuclear morphology with C . D and E : Comparison of apoptotic and necrotic thymus stained by TUNEL. D : Apoptotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). E : Necrotic thymus (green fluorescence, TUNEL staining; blue fluorescence, DAPI staining). Insets show high-magnification images of apoptotic and necrotic nuclei labeled by TUNEL (side of the inset , 80 μm). Strong positive staining is present in both cases. However necrotic thymus is uniformly positive with the loss of the characteristic pattern of cell death seen in glucocorticoid-induced apoptosis. F and G : Comparison of apoptotic and necrotic thymus using in situ ligation with blunt-ended probes detecting double-strand DNA breaks bearing 5′ phosphates. F : Apoptotic thymus, blunt ends detection. G : Necrotic thymus, blunt ends detection. Note that in situ ligation does not detect double-strand DNA breaks bearing 5′ phosphates in necrotic thymus. H and I : DNA nicks relegation in necrotic thymus using T4 DNA ligase. H : Necrotic thymus TUNEL-stained before T4 ligase pretreatment; I : the same thymus after T4 ligase pretreatment. DNA nicks do not contribute to the strong positive staining of necrotic thymus by TUNEL. No change in TUNEL signal intensity occurred after treatment of necrotic tissue with T4 DNA ligase to seal DNA nicks. Apoptosis was induced in the thymus by an intraperitoneal injection of 6 mg/kg of dexamethasone. Necrosis was induced in the thymus by freezing with liquid nitrogen. Scale bars: 200 μm ( C , E ); 300 μm ( G ); 150 μm ( I ).

    Article Snippet: The buffer was aspirated, and the full ligation mix containing the ligation buffer with the hairpin probe (35 μg/ml) and T4 DNA ligase (250 U/ml, Roche Molecular Biochemicals) was applied to the sections.

    Techniques: Staining, Fluorescence, In Situ, Ligation, TUNEL Assay, Labeling, Injection

    Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with T4 DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.

    Journal: Molecular and Cellular Biology

    Article Title: Trf4 and Trf5 Proteins of Saccharomyces cerevisiae Exhibit Poly(A) RNA Polymerase Activity but No DNA Polymerase Activity

    doi: 10.1128/MCB.25.22.10183-10189.2005

    Figure Lengend Snippet: Trf4 and Trf5 exhibit a poly(A) polymerase activity but no DNA polymerase activity. (A) DNA polymerase assays on a 29/75-nt primer/template partial duplex DNA substrate. The 29-nt primer was 32 P labeled at the 5′ end and annealed to a 75-nt template. Wild-type (wt) Trf4 (lane 3), Trf4 DD236,238AA (lane 4), and wild-type Trf5 (lane 5) (10 nM each) were incubated with the DNA substrate (20 nM) in the presence of each of the four dNTPs (100 μM). For positive and negative controls, parallel reactions were also carried out with T4 DNA polymerase (lane 2), and no added protein (NP) (lane 1). Reaction products were analyzed on a 15% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager. (B) DNA polymerase assays on an oligo(dT)/poly(dA) DNA substrate. A mixture of 12- to 18-nt oligo(dT) primers was 5′ end labeled and annealed to poly(dA) template. DNA polymerase reactions were carried out as described for panel A. (C) Poly(A) polymerase assays on a poly(A) substrate. Trf4 (lane 2), Trf4 DD236,238AA (lane 3), and Trf5 (lane 4) (10 nM each) were incubated with poly(A) RNA and [α- 32 P]ATP (50 μM) in the presence of 5 mM Mg 2+ and 0.5 mM Mn 2+ . A control reaction (lane 1) included no added protein (NP). Reaction products were resolved on a 15% polyacrylamide gel containing 8 M urea followed by PhosphorImager analyses of the incorporation of AMP into RNA tails. The sizes of the reaction products (in nucleotides) are indicated to the right of the gel.

    Article Snippet: The T4 DNA polymerase was purchased from Roche (Indianapolis, IN).

    Techniques: Activity Assay, Labeling, Incubation