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

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

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

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

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

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

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

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

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: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation

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: Five μL of T4 DNA ligase/ferromagnetic particle hybrid-dispersed solution, 2 μL of T4 DNA ligase buffer (Takara Bio Inc.), which consisted of 660 mM Tris-HCl (pH 7.6), 66 mM MgCl2 , 100 mM DTT and 1 mM ATP, 5 μL of aqueous solution containing 0.4 mM each of the DNA fragments, and 8 μL of sterilized water were mixed in a test tube, which was placed in a cylindrical container filled with circulating water, the temperature of which was regulated at 16 °C, from a constant-temperature bath (LTB-400, AS ONE CO.).

Techniques: DNA Ligation, Ligation

Journal: PLoS ONE

Article Title: High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

doi: 10.1371/journal.pone.0156106

Figure Lengend Snippet: pAK-TAG expression vector and high level expression of recombinant AK fusion proteins in soluble form. (A) Schematic representation of the pAK-TAG vector. (B) SDS-PAGE analysis of the expression of AK-TNFα, AK-TRAIL, and AK-T4 DNA ligase.

Article Snippet: Chemicals T4 DNA ligase, Taq polymerase, and restriction enzymes were obtained from Takara.

Techniques: Expressing, Plasmid Preparation, Recombinant, SDS Page

Journal: Nucleic Acids Research

Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

doi: 10.1093/nar/gkw998

Figure Lengend Snippet: RecA enhances T4 DNA ligase-catalyzed cohesive end- and blunt end-ligation. ( A ) Effects of the amounts of ligase in the presence or absence of RecA. Linear dsDNA with 3′-TGCA four nucleotide overhangs (panel (i)) or 5′-AGCT four nucleotide overhangs (panel (ii)) was incubated with the indicated amounts of T4 DNA ligase, in the presence or absence of RecA, for 40 min in the system without NAD but including ATP. In lanes M, DNA size and marker amounts are the same as in Figure 1B . The plots in panel (iii) show the fractions of residual substrate dsDNA (1L) against the amounts of T4 DNA ligase, quantified from at least three independent experiments. • (closed circles), dsDNA with 3′-four-nucleotide overhangs with RecA; ∘ (open circles), dsDNA with 3′ four-nucleotide overhangs without RecA; ▴ (closed triangles), dsDNA with 5′ four-nucleotide overhangs with RecA; Δ (open triangles), dsDNA with 5′-four-nucleotide overhangs without RecA. ( B ) The effects of RecA on the blunt end-ligation by T4 DNA ligase. Linear dsDNA with blunt ends was incubated with the indicated amounts of T4 DNA ligase, as in A, for 40 min, and the gel profile from a representative experiment is shown in panel (i). In lane M, DNA sizes and marker amounts are the same as in Figure 1B . The plot in panel (ii) was quantified from three independent experiments. ▪ (closed squares), dsDNA with blunt ends with RecA; □ (Open squares), dsDNA with blunt ends without RecA. It should be noted that T4 DNA ligase requires ATP as an essential cofactor, and thus the effects of nucleotide cofactors on the enhancement by RecA could not be tested.

Article Snippet: Escherchia coli and T4 DNA ligases were purchased from Takara Bio Company, Japan.

Techniques: Ligation, Incubation, Marker

Journal: Scientific Reports

Article Title: Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus

doi: 10.1038/srep05043

Figure Lengend Snippet: TALEN-induced genomic mutation in IL2RG . (a) T7 endonuclease I assay using TALENs for Jurkat cells. Jurkat cells were transfected with TALEN expression vectors by electroporation. After 5 days culture, genomic DNA was isolated and the TALEN target locus was amplified by PCR. A T7 endonuclease I assay was performed using purified PCR products. The arrowhead indicates the expected position of the digested products in the agarose gel. (b) Sequencing results of the PCR fragments, revealing different mutations in the TALEN target site. Jurkat cells were cultured for 5 days after electroporation, and cloning was performed by limiting dilution. Genomic DNA was isolated from cloned Jurkat cells and DNA sequencing was performed. Sequences for wild-type (WT) and deletion mutants (del1–4) are shown. (c) Functional analysis of genome-modified Jurkat cells. The level of IL2RG expression in genome-modified Jurkat cells was analyzed using flow cytometry. Cells were incubated with APC-conjugated-anti-hCD132 antibody for IL2RG and APC-IgG2b antibody as an isotype control. MFI, Mean Fluorescence Intensity of CD132. (d) qPCR analysis of BCL2 . BCL2 expression was examined 48 hr after the PMA and ionomycin stimulation in the presence of exogenous IL-2. Data are shown as mean ± SD (n = 3).

Article Snippet: The pVenus vector was used as the backbone to construct the IL2RG -targeting vector pVenus-L. A 5662 bp 5′ homology arm and a 3000 bp 3′ homology arm were amplified by PCR from the Jurkat cell genome, and cloned into the pVenus by using a DNA Ligation Kit.

Techniques: Mutagenesis, T7EI Assay, TALENs, Transfection, Expressing, Electroporation, Isolation, Amplification, Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Sequencing, Cell Culture, Clone Assay, DNA Sequencing, Functional Assay, Modification, Flow Cytometry, Cytometry, Incubation, Fluorescence, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Transcription activator-like effector nuclease-mediated transduction of exogenous gene into IL2RG locus

doi: 10.1038/srep05043

Figure Lengend Snippet: TALEN-mediated genome editing. (a) Top : Schematic of the endogenous IL2RG locus. Hind III, Hind III restriction sites used for Southern blot analysis; TM, transmembrane domain. Middle : Schematic of the targeting vector. The targeting vector contained Venus cDNA, lox-P-flanked Neomycin resistance cassette, and DT-A negative selectable marker. The 5′ homology arm upstream of the IL2RG start codon was cloned upstream of Venus cDNA, and the 3′ homology arm downstream of the IL2RG transmembrane sequence (exon 6) was cloned downstream of the Neomycin resistance cassette (Neor). 5′ probe, Probe used for Southern blot analysis. Bottom : Schematic of the targeted IL2RG locus. A novel Hind III restriction site would introduced when the targeted knock-in was successful. (b) Flow cytometric analysis of Venus in Jurkat cells with targeted knock-in of IL2RG . Each Jurkat cell clone was analyzed for YFP fluorescence expressed from knocked-in Venus cDNA. WT, wild-type Jurkat cells; KI 1-4, individual clones with targeted knock-in; KI 5, Venus hi ; MFI, Mean Fluorescence Intensity of Venus. (c) Southern blot analysis of Jurkat cells with targeted knock-in of IL2RG . Hind III digestion resulted in a 3788 bp band from the WT endogenous IL2RG locus and a 3515 bp band (containing Venus cDNA and the Neomycin resistance cassette) from the targeted knock-in. Targeting vector was used as positive control for targeted knock-in, and genomic DNA isolated from WT Jurkat cells was used as negative control.

Article Snippet: The pVenus vector was used as the backbone to construct the IL2RG -targeting vector pVenus-L. A 5662 bp 5′ homology arm and a 3000 bp 3′ homology arm were amplified by PCR from the Jurkat cell genome, and cloned into the pVenus by using a DNA Ligation Kit.

Techniques: Southern Blot, Plasmid Preparation, Marker, Clone Assay, Sequencing, Knock-In, Flow Cytometry, Fluorescence, Positive Control, Isolation, Negative Control