t4 dna ligase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 dna ligase
    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 Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t4 dna ligase - by Bioz Stars, 2020-01
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    Images

    1) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    2) Product Images from "Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039251

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    3) Product Images from "A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage"

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq756

    Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.
    Figure Legend Snippet: Principle, reaction efficiencies and NMR evidence for isotope labeling of each stem-loop of the RsmZ RNA separately. ( a ) Sequence-specific RNase H cleavages to obtain all four isotopically labeled stem-loop fragments. The yields of the cleavage reactions before HPLC purification are indicated, the values in brackets are expressing the yield after purification. The different stem-loops are colored (SL1: magenta, SL2: green, SL3: orange, SL4: cyan). ( b ) Splinted T4 DNA ligase mediated ligations of isotope labeled (in color) and unlabeled (in black) fragments. The unlabeled fragments were obtained in a similar way as the labeled fragments. ( c ) NMR evidence for the successful segmental isotope labeling of each stem-loop separately. 1 H- 15 N-HSQC NMR spectrum of the uniformly 15 N-labeled RsmZ RNA (left) and overlay of the 1 H- 15 N-HSQC NMR spectra of the four segmentally labeled RsmZ RNAs with each stem-loop labeled separately (right). The spectra were recorded on a Bruker 600 MHz spectrometer at 10°C.

    Techniques Used: Nuclear Magnetic Resonance, Labeling, Sequencing, High Performance Liquid Chromatography, Purification, Expressing

    4) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    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.
    Figure Legend 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.

    Techniques Used: Plasmid Preparation, Construct

    5) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    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.
    Figure Legend 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.

    Techniques Used: Plasmid Preparation, Construct

    6) Product Images from "FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis"

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

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2019.4355

    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.
    Figure Legend 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.

    Techniques Used: Plasmid Preparation, Construct

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    Article Snippet: .. HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). ..

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. Positive clones were further confirmed by DNA sequencing using an ABI capillary Genetic Analyzer 16.

    Amplification:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: The FSH1 forward inference sequence was amplified from the plasmid containing the full-length cDNA sequence of the FSH1 gene with the FSH1 primers presented in . .. 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.).

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: These primers amplified the last 115 bp of exon 1, intron 1, exon 2, intron 2 and the first 157 bp of exon 3, using Platinum® Taq DNA Polymerase High Fidelity (Invitrogen). .. The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen).

    Mass Spectrometry:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: .. A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Positive Control:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: The phosphorylation of oligo 11 by T4 DNA ligase was performed in 25 µl of phosphorylation mixture containing 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 10 mM DTT, 0.6 µCi/µl of [γ-32P] ATP, 4 µM of oligo 11, and 0.5 Weiss units/µl of T4 DNA ligase (Fermentas). .. The positive control was oligo 11 phosphorylated by T4 PNK.

    Synthesized:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: The pUC-PUT plasmid was synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd. A map of each construct is presented in . .. 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.).

    Construct:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: To construct the final FSH1 double-stranded RNA interference (dsRNAi) plasmid pCB309-PFUFT, three steps of ligation were performed as follows. .. 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.).

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT. .. Intermediate and final vectors were propagated in E. coli DH5α cells (Beijing Huayueyang Biotechnology Co., Ltd.), purified by plasmid mini-preparations (Nucleospin Plasmid kit; Macherey-Nagel GmbH and Co. KG) and confirmed by a series of restriction enzyme digestions, followed by 1% agarose gel electrophoresis.

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen). .. Potential splicing factor binding sites within the Mcl-1 minigene construct were mutated using the Q5 site-directed mutagenesis kit (New England BioLabs) with forward primer 5′-AAGTATCACAAAAGTTCTCGTAAGG-3′, and reverse primer 5′-TCTGCTAATGGTTCGATG-3′ for SRSF1 binding site, and forward primer 5′-GGATGAAAGGCGCCTTGGAGTG-3′, and reverse primer 5′-TTAAGGCAAACTTACCCAG-3′ for hnRNP F/H binding site.

    Article Title: Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer
    Article Snippet: .. HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). ..

    Electrophoresis:

    Article Title: Application of linker technique to trap transiently interacting protein complexes for structural studies
    Article Snippet: ✓ TAE buffer: Tris-acetate-EDTA buffer is used for preparing agarose gels and for electrophoresis. .. ✓ T4 DNA ligase: Double digested vector and insert were ligated using T4 DNA ligase (Fermentas) using manufacturer’s instructions.

    Incubation:

    Article Title: Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis
    Article Snippet: .. DNA fragments were self-ligated by T4 DNA ligase, and the reaction was carried out in a final volume of 20 μl containing 0.5 μl digested DNA fragments, 2 μl 10× buffer, 0.5 μl T4 DNA ligase (Thermo), 17 μl ddH2 O, incubated for 12 h at 10°C. .. Nest-PCR was performed in this experiment, 0.5 μl ligated production was used as PCR template, the specific primers used in the first PCR reaction were 201RF1 and 201RR1, products from first PCR reaction were diluted to 1/1000 as the template for the second PCR reaction, and specific primer 201RF2 and 201RR2 were used.

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: .. For ligation with T4 DNA ligase, the duplex was mixed with 0.1 U/μl T4 DNA ligase (Fermentas), 10 mM Tris–Acetate pH 7.5, 10 mM MgAc2 , 50 mM KAc, 1 mM ATP (Fermentas) and 0.1 μg/μl BSA for 2 min at 75°C, 1 min at 45°C, 2 min at 30°C, 30 min at 37°C and finally 10 min at 65°C (the T4 DNA ligase was added prior to the incubation at 37°C). ..

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: When T4 DNA ligase was compared to Ampligase, 0.1 U/μl of T4 DNA ligase (Thermo Scientific) in 1X T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP) supplemented with 0.2 μg/μl BSA, 200 nM ATP and 200 mM NaCl was used to ligate 0.1 μM of the padlock gap probe and 2 μM of each gap probe for 30 min at 37°C. .. After ligation the slides for mitochondrial DNA detection were incubated 5 min at 37°C in 2x SSC, 0.05% Tween-20, and rinsed in wash buffer to remove non-hybridized probes.

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: Meanwhile, one μg of the pSilencer 3.0-H1 was cut with restriction enzymes Bam HI and Hin dIII (New England BioLabs, Ipswich, MA, USA), then incubated at 37 °C for 4 h. On the other hand, pmirGLO was cut with Nhe I and Xho I (New England BioLabs, Ipswich, MA, USA), followed by incubation at 37 °C for 4 h. For pmirGLO, the plasmids were treated with 1 µL of Antarctic phosphatase (New England BioLabs, Ipswich, MA, USA). .. After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol.

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: Because an available phosphate group on the 5′-end of the ligation substrate is an absolute requirement for enzymatic ligation reactions, the padlock gap probe and the gap probes were phosphorylated with 0.2 U/μl of T4 PNK enzyme (Thermo Scientific) in supplied buffer A and 1 mM ATP at 37°C for 30 min, followed by inactivation of the enzyme at 65°C for 10 min. Hybridization and ligation were performed in a single reaction containing 0.1 μM of the padlock gap probe and 2 μM of each gap probe (1 μM when 7 gap probes were used to test FFPE samples) using 0.5 U/μl of Ampligase (Epicentre) in 1x Ampligase buffer (20 mM Tris-HCl pH 8.3, 25 mM KCl, 10 mM MgCl2 , 0.5 mM NAD and 0.01% Triton X-100), 0.2 μg/μl BSA and 0,05 mM KCl (125 mM KCl was used for mtDNA detection) at 45°C for 60 min. .. When T4 DNA ligase was compared to Ampligase, 0.1 U/μl of T4 DNA ligase (Thermo Scientific) in 1X T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP) supplemented with 0.2 μg/μl BSA, 200 nM ATP and 200 mM NaCl was used to ligate 0.1 μM of the padlock gap probe and 2 μM of each gap probe for 30 min at 37°C.

    Activity Assay:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Expressing:

    Article Title: Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer
    Article Snippet: .. HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). ..

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: Plasmid Construction pSilencer 3.0-H1 (Ambion, Austin, TX, USA) and pmirGLO (Promega, Madison, WI, USA) were used as a vector backbone to produce microRNA expression vectors and reporter vectors, respectively. .. After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol.

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Kinase Assay:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: Paragraph title: Kinase Assay for T4 DNA Ligase ... The phosphorylation of oligo 11 by T4 DNA ligase was performed in 25 µl of phosphorylation mixture containing 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 10 mM DTT, 0.6 µCi/µl of [γ-32P] ATP, 4 µM of oligo 11, and 0.5 Weiss units/µl of T4 DNA ligase (Fermentas).

    Transformation Assay:

    Article Title: Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer
    Article Snippet: HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). .. MAX efficiency competent cells (DH5-alpha; Thermofisher) were transformed with ligation mixture, and plated on LB-ampicillin agar at 37 °C overnight.

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol. .. The plasmids were transformed into E. coli strain JM109 competent cells (RBC Bioscience, New Taipei City, Taiwan) by heat shock method.

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Hybridization:

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: Paragraph title: Padlock gap probe hybridization and gap probe ligation ... When T4 DNA ligase was compared to Ampligase, 0.1 U/μl of T4 DNA ligase (Thermo Scientific) in 1X T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP) supplemented with 0.2 μg/μl BSA, 200 nM ATP and 200 mM NaCl was used to ligate 0.1 μM of the padlock gap probe and 2 μM of each gap probe for 30 min at 37°C.

    High Performance Liquid Chromatography:

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    DNA Ligation:

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: .. When T4 DNA ligase was compared to Ampligase, 0.1 U/μl of T4 DNA ligase (Thermo Scientific) in 1X T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP) supplemented with 0.2 μg/μl BSA, 200 nM ATP and 200 mM NaCl was used to ligate 0.1 μM of the padlock gap probe and 2 μM of each gap probe for 30 min at 37°C. .. After ligation the slides for mitochondrial DNA detection were incubated 5 min at 37°C in 2x SSC, 0.05% Tween-20, and rinsed in wash buffer to remove non-hybridized probes.

    Ligation:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: To construct the final FSH1 double-stranded RNA interference (dsRNAi) plasmid pCB309-PFUFT, three steps of ligation were performed as follows. .. 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.).

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: .. The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen). .. Potential splicing factor binding sites within the Mcl-1 minigene construct were mutated using the Q5 site-directed mutagenesis kit (New England BioLabs) with forward primer 5′-AAGTATCACAAAAGTTCTCGTAAGG-3′, and reverse primer 5′-TCTGCTAATGGTTCGATG-3′ for SRSF1 binding site, and forward primer 5′-GGATGAAAGGCGCCTTGGAGTG-3′, and reverse primer 5′-TTAAGGCAAACTTACCCAG-3′ for hnRNP F/H binding site.

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: Kinase Assay for T4 DNA Ligase To explore the ligation mechanism of DNA linkers with 5′-OH ends, oligo 11 of linker F were phosphorylated by using [γ-32P] ATP and T4 DNA ligase. .. The phosphorylation of oligo 11 by T4 DNA ligase was performed in 25 µl of phosphorylation mixture containing 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 10 mM DTT, 0.6 µCi/µl of [γ-32P] ATP, 4 µM of oligo 11, and 0.5 Weiss units/µl of T4 DNA ligase (Fermentas).

    Article Title: Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer
    Article Snippet: HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). .. MAX efficiency competent cells (DH5-alpha; Thermofisher) were transformed with ligation mixture, and plated on LB-ampicillin agar at 37 °C overnight.

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: .. For ligation with T4 DNA ligase, the duplex was mixed with 0.1 U/μl T4 DNA ligase (Fermentas), 10 mM Tris–Acetate pH 7.5, 10 mM MgAc2 , 50 mM KAc, 1 mM ATP (Fermentas) and 0.1 μg/μl BSA for 2 min at 75°C, 1 min at 45°C, 2 min at 30°C, 30 min at 37°C and finally 10 min at 65°C (the T4 DNA ligase was added prior to the incubation at 37°C). ..

    Article Title: Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
    Article Snippet: Paragraph title: Padlock gap probe hybridization and gap probe ligation ... When T4 DNA ligase was compared to Ampligase, 0.1 U/μl of T4 DNA ligase (Thermo Scientific) in 1X T4 DNA ligation buffer (40 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, 0.5 mM ATP) supplemented with 0.2 μg/μl BSA, 200 nM ATP and 200 mM NaCl was used to ligate 0.1 μM of the padlock gap probe and 2 μM of each gap probe for 30 min at 37°C.

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: Each 10 μL of the top- and bottom-strand oligonucleotides (10 nM) was added with 5 μL of 5× rapid ligation buffer (Thermo Scientific, Vilnius, Lithuania), then denatured at 90 °C for 5 min, followed by annealing at 25 °C for 1 h ( ). .. After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol.

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Generated:

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: The Mcl-1 minigene was generated from human genomic DNA using specific Mcl-1 PCR primers containing specific restriction sites ( ): forward primer 5′-TCTAGAGGCGCCAAGGACACAAA-3′ (XbaI), reverse primer 5′-AAGCTTGGTGGTTGGTTAAAAGTCAACTATTG-3′ (HindIII). .. The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen).

    DNA Sequencing:

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. Positive clones were further confirmed by DNA sequencing using an ABI capillary Genetic Analyzer 16.

    Sequencing:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: The FSH1 forward inference sequence was amplified from the plasmid containing the full-length cDNA sequence of the FSH1 gene with the FSH1 primers presented in . .. 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.).

    Article Title: Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis
    Article Snippet: The gene-specific primers 201F and 201R were designed to amplify the partial DNA fragment of Fm201 according to the available sequence of Fm201 EST. .. DNA fragments were self-ligated by T4 DNA ligase, and the reaction was carried out in a final volume of 20 μl containing 0.5 μl digested DNA fragments, 2 μl 10× buffer, 0.5 μl T4 DNA ligase (Thermo), 17 μl ddH2 O, incubated for 12 h at 10°C.

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Binding Assay:

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen). .. Potential splicing factor binding sites within the Mcl-1 minigene construct were mutated using the Q5 site-directed mutagenesis kit (New England BioLabs) with forward primer 5′-AAGTATCACAAAAGTTCTCGTAAGG-3′, and reverse primer 5′-TCTGCTAATGGTTCGATG-3′ for SRSF1 binding site, and forward primer 5′-GGATGAAAGGCGCCTTGGAGTG-3′, and reverse primer 5′-TTAAGGCAAACTTACCCAG-3′ for hnRNP F/H binding site.

    Mutagenesis:

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen). .. Potential splicing factor binding sites within the Mcl-1 minigene construct were mutated using the Q5 site-directed mutagenesis kit (New England BioLabs) with forward primer 5′-AAGTATCACAAAAGTTCTCGTAAGG-3′, and reverse primer 5′-TCTGCTAATGGTTCGATG-3′ for SRSF1 binding site, and forward primer 5′-GGATGAAAGGCGCCTTGGAGTG-3′, and reverse primer 5′-TTAAGGCAAACTTACCCAG-3′ for hnRNP F/H binding site.

    Size-exclusion Chromatography:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: The PCR mixture was as follows: 1.25U LA Taq (Takara Bio, Inc.), 1 µ l FSH1 plasmid (20 ng/µ l), 1 µ l FSH1-F primer, 1 µ l FSH1-R primer, 5 µ l 10X LA Taq Buffer, 4 µ l dNTP (2.5 mM), and ddH2 O up to a total volume of 50 µ l. The thermocycling conditions were as follows: Initial denaturation at 94°C for 1 min; followed by 30 cycles of 94°C for 30 sec, 55°C for 45 sec and 72°C for 30 sec, and a final extension 72°C for 10 min. PCR products were gel purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.). .. 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.).

    Labeling:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: The kinase assay for E. coli DNA ligase was not performed because NAD+ labeled with 32P was not available for us. .. The phosphorylation of oligo 11 by T4 DNA ligase was performed in 25 µl of phosphorylation mixture containing 40 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 10 mM DTT, 0.6 µCi/µl of [γ-32P] ATP, 4 µM of oligo 11, and 0.5 Weiss units/µl of T4 DNA ligase (Fermentas).

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: 5′-Ligation of oligonucleotide after MutY glycosylase and Endonuclease IV treatment The tracking oligo was created by adding 15 nM of the labeled track_relig1 with 15 nM track_relig2 and hybridizing to 15 nM probe_relig in a mixture of 0.2 U/μl Ampligase (Epicentre), 20 mM Tris–HCl pH 8.3, 25 mM KCl, 10 mM MgCl2 , 0.5 mM NAD, 0.01 % Triton X-100 and 0.1 μg/μl BSA for 2 min at 75°C, 1 min at 45°C, 2 min at 30°C and finally 2 min at 45°C. .. For ligation with T4 DNA ligase, the duplex was mixed with 0.1 U/μl T4 DNA ligase (Fermentas), 10 mM Tris–Acetate pH 7.5, 10 mM MgAc2 , 50 mM KAc, 1 mM ATP (Fermentas) and 0.1 μg/μl BSA for 2 min at 75°C, 1 min at 45°C, 2 min at 30°C, 30 min at 37°C and finally 10 min at 65°C (the T4 DNA ligase was added prior to the incubation at 37°C).

    Purification:

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

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT. .. Intermediate and final vectors were propagated in E. coli DH5α cells (Beijing Huayueyang Biotechnology Co., Ltd.), purified by plasmid mini-preparations (Nucleospin Plasmid kit; Macherey-Nagel GmbH and Co. KG) and confirmed by a series of restriction enzyme digestions, followed by 1% agarose gel electrophoresis.

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    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.). .. The FSH1 gene was then ligated into pUC-PFUT following digestion with Bgl II and Kpn I and ligated by T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) and this was called pUC-PFUFT.

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Article Title: Application of linker technique to trap transiently interacting protein complexes for structural studies
    Article Snippet: ✓ T4 DNA ligase: Double digested vector and insert were ligated using T4 DNA ligase (Fermentas) using manufacturer’s instructions. .. ✓ Gel extraction kit: PCR products were purified using the GeneAll kit (GeneAll Biotechnology, Korea).

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Polymerase Chain Reaction:

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

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    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.). .. Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT.

    Article Title: Arbuscular Mycorrhizal Fungal 14-3-3 Proteins Are Involved in Arbuscule Formation and Responses to Abiotic Stresses During AM Symbiosis
    Article Snippet: Reverse PCR was utilized to get the 5′ flanking sequence of Fm201 gene. .. DNA fragments were self-ligated by T4 DNA ligase, and the reaction was carried out in a final volume of 20 μl containing 0.5 μl digested DNA fragments, 2 μl 10× buffer, 0.5 μl T4 DNA ligase (Thermo), 17 μl ddH2 O, incubated for 12 h at 10°C.

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: The PCR product was cloned into the TOPO®-TA cloning vector (Invitrogen) and sequenced. .. The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen).

    Article Title: Application of linker technique to trap transiently interacting protein complexes for structural studies
    Article Snippet: ✓ T4 DNA ligase: Double digested vector and insert were ligated using T4 DNA ligase (Fermentas) using manufacturer’s instructions. .. ✓ Gel extraction kit: PCR products were purified using the GeneAll kit (GeneAll Biotechnology, Korea).

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Plasmid Preparation:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: Paragraph title: Construction of pCB309-PFUFT-FSH1 knockdown vector ... 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.).

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT. .. Intermediate and final vectors were propagated in E. coli DH5α cells (Beijing Huayueyang Biotechnology Co., Ltd.), purified by plasmid mini-preparations (Nucleospin Plasmid kit; Macherey-Nagel GmbH and Co. KG) and confirmed by a series of restriction enzyme digestions, followed by 1% agarose gel electrophoresis.

    Article Title: Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells
    Article Snippet: Paragraph title: Mcl-1 Minigene Plasmid Construction ... The fragment was then subcloned into pcDNA3.1 by restriction digestion and ligation using T4 DNA ligase (Invitrogen).

    Article Title: Acetylation of conserved DVL-1 lysines regulates its nuclear translocation and binding to gene promoters in triple-negative breast cancer
    Article Snippet: .. HA-hDVL-1 pcDNA3.1 (+) construct generation The full-length 2.1-kb DVL-1 gene was cloned into KpnI/EcoRI restriction enzyme sites of 5.4-kb pcDNA3.1(+) mammalian expression plasmid using T4 DNA ligase (Invitrogen). ..

    Article Title: Application of linker technique to trap transiently interacting protein complexes for structural studies
    Article Snippet: .. ✓ T4 DNA ligase: Double digested vector and insert were ligated using T4 DNA ligase (Fermentas) using manufacturer’s instructions. .. ✓ Gel extraction kit: PCR products were purified using the GeneAll kit (GeneAll Biotechnology, Korea).

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: Paragraph title: 2.6. Plasmid Construction ... After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol.

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α. .. The transformants were screened by colony PCR using T7 promoter and T7 terminator primers and DreamTaq polymerase (Thermo Scientific).

    Recombinant:

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: Paragraph title: Recombinant plasmid construction. ... Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α.

    Agarose Gel Electrophoresis:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT. .. Intermediate and final vectors were propagated in E. coli DH5α cells (Beijing Huayueyang Biotechnology Co., Ltd.), purified by plasmid mini-preparations (Nucleospin Plasmid kit; Macherey-Nagel GmbH and Co. KG) and confirmed by a series of restriction enzyme digestions, followed by 1% agarose gel electrophoresis.

    Produced:

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: When these manufacturers were questioned, they stated that their T4 DNA ligases had very high quality and it was very unlikely that there would be PNK in their ligases because their ligases were produced by using E. coli cells and production lines that were different from those for T4 PNK. .. A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells .

    Concentration Assay:

    Article Title: A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage
    Article Snippet: .. A typical large-scale ligation reaction using T4 DNA ligase was 10 μM in RNA fragments, 15 μM in DNA splint oligo, 10% PEG-4000 in 40 mM Tris–HCl pH = 7.8, 0.5 mM ATP, 10 mM MgCl2 , 10 mM DTT using 50 U T4 DNA ligase (fermentas) per nmol of RNA to be ligated or 2 μM final concentration of in-house produced T4 DNA ligase. .. The reactions were subjected to HPLC purification followed by n -butanol extraction and lyophilization.

    Article Title: High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses
    Article Snippet: After that, the annealed fragment was ligated into linearized pSilencer3.0-H1 or pmirGLO with T4 DNA ligase (Thermo Scientific, Vilnius, Lithuania), according to the manufacturer’s protocol. .. The concentration of each plasmid was measured by NanoPhotometer® (Implen, Munich, Germany).

    DNA Purification:

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: The PCR mixture was as follows: 1.25U LA Taq (Takara Bio, Inc.), 1 µ l FSH1 plasmid (20 ng/µ l), 1 µ l FSH1-F primer, 1 µ l FSH1-R primer, 5 µ l 10X LA Taq Buffer, 4 µ l dNTP (2.5 mM), and ddH2 O up to a total volume of 50 µ l. The thermocycling conditions were as follows: Initial denaturation at 94°C for 1 min; followed by 30 cycles of 94°C for 30 sec, 55°C for 45 sec and 72°C for 30 sec, and a final extension 72°C for 10 min. PCR products were gel purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.). .. 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.).

    Article Title: FSH1 regulates the phenotype and pathogenicity of the pathogenic dermatophyte Microsporum canis
    Article Snippet: .. Finally, the two plasmids, pUC-PFUFT and pCB309 were digested using Spe I and Sac I, purified using a Universal DNA Purification kit (Tiangen Biotech Co., Ltd.) and ligated using T4 DNA ligase (Invitrogen; Thermo Fisher Scientific, Inc.) to construct the final FSH1 dsRNAi plasmid, pCB309-PFUFT. .. Intermediate and final vectors were propagated in E. coli DH5α cells (Beijing Huayueyang Biotechnology Co., Ltd.), purified by plasmid mini-preparations (Nucleospin Plasmid kit; Macherey-Nagel GmbH and Co. KG) and confirmed by a series of restriction enzyme digestions, followed by 1% agarose gel electrophoresis.

    Gel Extraction:

    Article Title: Application of linker technique to trap transiently interacting protein complexes for structural studies
    Article Snippet: ✓ T4 DNA ligase: Double digested vector and insert were ligated using T4 DNA ligase (Fermentas) using manufacturer’s instructions. .. ✓ Gel extraction kit: PCR products were purified using the GeneAll kit (GeneAll Biotechnology, Korea).

    Article Title: Structure Elucidation and Biochemical Characterization of Environmentally Relevant Novel Extradiol Dioxygenases Discovered by a Functional Metagenomics Approach
    Article Snippet: The PCR product was digested using NdeI and XhoI (Thermo Scientific) and purified using a QIAquick Gel Extraction kit (Qiagen). .. Purified PCR product was ligated into a predigested (NdeI/XhoI) pET28a vector with an N-terminal 6×His tag sequence using T4 DNA ligase (Thermo Scientific), and the reaction mixture was incubated at 22°C for 1 h. The ligation product was transformed into Escherichia coli DH5α.

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    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1134 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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