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    New England Biolabs cutsmart
    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within <t>CutSmart</t> buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.
    Cutsmart, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Journal: Nucleic Acids Research

    Article Title: Exonuclease combinations reduce noises in 3D genomics technologies

    doi: 10.1093/nar/gkaa106

    Figure Lengend Snippet: Experimental results for exonuclease combinations treatment. ( A ) The cleavage mechanism of Lamada and Exonuclease I combinations. Other exonuclease combinations (Lambda and RecJF; Exonuclease I and Exonuclease III) are shown in Supplementary Figure S1A and Supplementary Table S1 . ( B ) Three exonuclease combinations (LRL; LRC; LIC) removed linear DNA from a paradigm mixture. M, 1 kb DNA ladder; P, pGL4.23 plasmid; L, linearized plasmid; X, mixture (plasmid and linear DNA 1:1); 1-X, LRL-Mixture, LRL to cut mixture; 2-X, LRC-Mixture, LRC to cut mixture; 3-X, LIC-Mixture, LIC to cut mixture; 3-P, LIC to cut plasmid; 3-L, LIC-Lin, LIC to cut linearized plasmid; MS, supercoiled ladder. ( C ) I+III combination and Exonuclease VIII, truncated elimination tests. 5-X, VIII4-Mixture, Exonuclease VIII, truncated within Buffer 4 to remove mixture; 6-X, VIIIC-Mixture, Exonuclease VIII, truncated within CutSmart buffer to remove mixture; 4-X: I+III-Mixture, I+III to remove mixture. Loading samples for agarose gel electrophoresis were purified by phenol-chloroform.

    Article Snippet: Linearized plasmid DNA was obtained in the following 200 μl solution/per reaction, including 23.4 μl plasmid, 5 μl BamHI-HF (HindIII for P2) (NEB), 20 μl 10× Cutsmart Buffer (NEBuffer 2 for P2) and 151.6 μl H2 O.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Purification

    Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Exo I III versus Exo T5 digestion of plasmid DNA. (A) Diagrams showing expected digestion results of various plasmid DNA species. A break in the circle denotes the nick on the DNA strand. (B and C) Plasmid pCI-HBc (2.5 ng) was mixed with 20 μl of mock PF DNA extracted from uninduced HepAD38 cells. The DNA mix was first treated with Nb.BbvCI (5 units) to nick the plasmid DNA specifically on the minus strand (B and C, lanes 5 to 8) or was left untreated (B and C, lanes 1 to 4) before digestion with Exo I III (5 units and 25 units, respectively) in two different buffers or with Exo T5 (5 units). The DNA samples were then resolved on an agarose gel, and HBc DNA was detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. B3, 1× NEB buffer 3; BCS, 1× NEB buffer Cutsmart; PE, phenol extraction.

    Article Snippet: For Exo I and Exo III (Exo I & III) digestion, 20 μl PF DNA prepared as described above was treated with 0.25 μl each of Exo I (NEB; 5 units) and Exo III (NEB; 25 units) in 1× NEB Cutsmart buffer (50 mM potassium acetate, 20 mM Tris–acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin [BSA], pH 7.9 [prepared at 25°C]) or 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 [prepared at 25°C]), as indicated, at 37°C for 2 to 3 h in a total volume of 23 μl.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Southern Blot, Migration

    Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Exo I III versus Exo T5 digestion of HBV core and PF DNA. (A) Diagrams showing expected results of digestion with various HBV PF DNA species. Left, structures of known and potential HBV PF DNA species; middle and right, expected digestion products of the various DNA species. The DNA species in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in the current study (see the text for details). The black dot at the 5′ end of the minus strand of the PF-RC and PF-DSL DNA denotes the unknown modification of this end upon removal of the RT protein (deproteination; see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (B) or PF DNA (20 μl) extracted from induced HepAD38 cells (C) was treated with Exo I III (5 units and 25 units, respectively) (lanes 3 and 10) or Exo T5 (5 units) (lanes 6 and 13) in 1× NEB CutSmart buffer. Subsequently, MfeI-HF (10 units) was used to linearize CCC DNA (lanes 5, 7, 12, and 14) and Exo T5 (5 units) was used to digest the SS circular DNA (lanes 4 and 11). Heat treatment (95°C, 10 min) was used to denature RC DNA to SS linear DNA (lanes 2 and 9). The DNA samples were then resolved on an agarose gel, and the various HBV DNA species were detected by Southern blotting using a riboprobe specific for the plus-strand (lanes 1 to 7) or minus-strand (lanes 8 to 14) DNA. The diagrams on the sides depict the various DNA species and their migration on the gel. The positions of the various RC DNA species, CCC DNA species, and SS linear and circular DNA species are indicated by the schematic diagrams. Note that the linearized CCC DNA comigrates with the DSL DNA, a minor form present in both core DNA and PF DNA (lanes 1 and 8).

    Article Snippet: For Exo I and Exo III (Exo I & III) digestion, 20 μl PF DNA prepared as described above was treated with 0.25 μl each of Exo I (NEB; 5 units) and Exo III (NEB; 25 units) in 1× NEB Cutsmart buffer (50 mM potassium acetate, 20 mM Tris–acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin [BSA], pH 7.9 [prepared at 25°C]) or 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 [prepared at 25°C]), as indicated, at 37°C for 2 to 3 h in a total volume of 23 μl.

    Techniques: Countercurrent Chromatography, Modification, Agarose Gel Electrophoresis, Southern Blot, Migration

    Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Journal: Journal of Virology

    Article Title: Identification of an Intermediate in Hepatitis B Virus Covalently Closed Circular (CCC) DNA Formation and Sensitive and Selective CCC DNA Detection

    doi: 10.1128/JVI.00539-17

    Figure Lengend Snippet: Confirmation of the closed circular minus strand in the processed RC DNA by BmgBI or Nt.BbvCI and Exo I III digestion. (A and D) Diagrams showing expected results of digestion performed with various HBV PF DNA species. The short line intersecting the circle denotes the site of BmgBI digestion (A) or Nt.BbvCI nicking (D). The presence of the RNA (short gray line) at the 5′ end of the plus strand in RC DNA prevents BmgBI digestion (panel A; arrow blocked by a short line). The black dot at the 5′ end of the minus strand of the PF-RC DNA denotes the unknown modification of this end upon removal of the RT protein. The DNA species indicated in the rectangular box, with a covalently closed minus strand and an open plus strand, represents a potential intermediate during RC DNA to CCC DNA conversion that was identified in this study (see the text for details). (B and C) HBV core DNA (0.3 μl) combined with mock PF DNA (20 μl) extracted from uninduced HepAD38 cells (lanes 1 to 3) or PF DNA (lanes 4 to 6) extracted from induced HepAD38 cells was treated with BmgBI (5 units) in 1× NEB buffer 3 to linearize all supercoiled and nicked CCC DNA (lanes 2, 3, 5, and 6) or was mock treated (lanes 1 and 4). For lanes 3 and 6, the DNA samples were further digested with Exo I III after BmgBI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (B) or minus-strand (C) DNA. The diagrams on the right of panel C depict the various DNA species and their migration on the gel. (E) PF DNA extracted from induced HepAD38 cells was treated with Nt.BbvCI (5 units) in 1× NEB Cutsmart buffer to nick all CCC DNA (lanes 3, 4, 7, and 8) or mock treated (lanes 1 and 5). For lanes 4 and 8, the DNA samples were further digested with Exo I III after Nt.BbvCI treatment. The samples were then resolved on an agarose gel, and various HBV DNA species were detected by Southern blotting using a riboprobe specific for the viral plus-strand (lanes 1 to 4) or minus-strand (lanes 5 to 8) DNA. The diagrams on the right depict the various DNA species and their migration on the gel. Marker, the DNA marker lane. The size of the DNA markers is indicated (in kilobase pairs). The blank spaces between the lanes in panels B, C, and E indicate where other lanes from the same gel that were deemed nonessential for this work were cropped out during the preparation of the figure.

    Article Snippet: For Exo I and Exo III (Exo I & III) digestion, 20 μl PF DNA prepared as described above was treated with 0.25 μl each of Exo I (NEB; 5 units) and Exo III (NEB; 25 units) in 1× NEB Cutsmart buffer (50 mM potassium acetate, 20 mM Tris–acetate, 10 mM magnesium acetate, 100 μg/ml bovine serum albumin [BSA], pH 7.9 [prepared at 25°C]) or 1× NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9 [prepared at 25°C]), as indicated, at 37°C for 2 to 3 h in a total volume of 23 μl.

    Techniques: Modification, Countercurrent Chromatography, Agarose Gel Electrophoresis, Southern Blot, Migration, Marker