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  • 99
    New England Biolabs puc19
    Puc19, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2021-06
    99/100 stars
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    86
    Thermo Fisher puc19
    Domain movements during Figure‐eight formation The position of helix ɑ9 B relative to the flanking DNA. TnpA is depicted in a surface charge representation. ID B movement associated with binding flanking DNA. The double‐headed white arrow shows the 8 Å shift between the positions of 3′‐terminal residues (in red) of R‐TIRs in the PRC and PCC. Normal mode analysis of CD and ID domains of IS Cth4 Tnp (residues 160–407) reveals the potential for relative motion (marked with black arrow field). Colors correspond to the crystallographic B‐factor of the model. Comparison of PCC and STC1 structures, backside view. Close‐up of cd B in STC1 structure with bound junction spacer (in orange) and the tip of the recipient L‐TIR (in gray). Labeled residue side chains shown as sticks belong to the cd B ‐binding site. Diagram of observed interactions between TnpA and tip of donor R‐TIR, junction spacer and recipient L‐TIR in STC1. Interactions with the rest of R‐TIR are identical to those in the PCC. In vitro integration of TIR junction mimics with modified L‐TIRs or spacer sequence. Lane 1, <t>pUC19</t> alone after 24 h of incubation in reaction buffer omitting only oligonucleotide substrate. White triangles mark the TIR ends. “M”, base pair marker. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.
    Puc19, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

    94
    TaKaRa puc19
    The SMR domain of SOT1 has nuclease activity. ( A ) Purified MBP, SOT1 SMR , and Gm-SOT1 SMR used for nuclease activity were resolved by SDS/PAGE with Coomassie Brilliant Blue (CBB) staining or with the analyses of immunoblot using the MBP antibody. The marker sizes are shown to the Left . ( B ) DNA endonuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR at different concentrations were incubated with 5 ng <t>pUC19</t> plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . The reactions were stopped by loading buffer and were electrophoresed on 1.2% (wt/vol) agarose gels. Parallel experiments were carried out with EcoRI to linearize pUC19 or with H 2 O as a control. OC, open circular; CCC, covalently closed-circular forms of pUC19. ( C ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on DNA nicking activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 5 ng pUC19 was incubated with H 2 O, MgCl 2 , CaCl 2 , or MnCl 2 and 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR . The concentration of each cation was 3 mM. ( D ) Analyses of DNA nicking activity of Gm-SOT1. MBP and Gm-SOT1 were incubated with 5 ng pUC19 plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . ( E ) RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( F ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR were incubated with total wild-type Arabidopsis RNA in the presence of MgCl 2 , CaCl 2 , or MnCl 2 . The concentration of each cation was 3 mM. ( G ) Accumulation of total RNAs in WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 plants. A total of 3 μg total RNAs from 12-d-old WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 seedlings were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( H ) Analyses of RNA nuclease activity of Gm-SOT1. MBP and Gm-SOT1 with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were detected by electrophoretic separation in agarose/formaldehyde gels with ethidium bromide staining.
    Puc19, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/puc19/product/TaKaRa
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    puc19 - by Bioz Stars, 2021-06
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    N/A
    Standard format Plasmid sent in bacteria as agar stab
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    N/A
    Standard format Plasmid sent in bacteria as agar stab
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    Image Search Results


    Domain movements during Figure‐eight formation The position of helix ɑ9 B relative to the flanking DNA. TnpA is depicted in a surface charge representation. ID B movement associated with binding flanking DNA. The double‐headed white arrow shows the 8 Å shift between the positions of 3′‐terminal residues (in red) of R‐TIRs in the PRC and PCC. Normal mode analysis of CD and ID domains of IS Cth4 Tnp (residues 160–407) reveals the potential for relative motion (marked with black arrow field). Colors correspond to the crystallographic B‐factor of the model. Comparison of PCC and STC1 structures, backside view. Close‐up of cd B in STC1 structure with bound junction spacer (in orange) and the tip of the recipient L‐TIR (in gray). Labeled residue side chains shown as sticks belong to the cd B ‐binding site. Diagram of observed interactions between TnpA and tip of donor R‐TIR, junction spacer and recipient L‐TIR in STC1. Interactions with the rest of R‐TIR are identical to those in the PCC. In vitro integration of TIR junction mimics with modified L‐TIRs or spacer sequence. Lane 1, pUC19 alone after 24 h of incubation in reaction buffer omitting only oligonucleotide substrate. White triangles mark the TIR ends. “M”, base pair marker. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Structures of ISCth4 transpososomes reveal the role of asymmetry in copy‐out/paste‐in DNA transposition

    doi: 10.15252/embj.2020105666

    Figure Lengend Snippet: Domain movements during Figure‐eight formation The position of helix ɑ9 B relative to the flanking DNA. TnpA is depicted in a surface charge representation. ID B movement associated with binding flanking DNA. The double‐headed white arrow shows the 8 Å shift between the positions of 3′‐terminal residues (in red) of R‐TIRs in the PRC and PCC. Normal mode analysis of CD and ID domains of IS Cth4 Tnp (residues 160–407) reveals the potential for relative motion (marked with black arrow field). Colors correspond to the crystallographic B‐factor of the model. Comparison of PCC and STC1 structures, backside view. Close‐up of cd B in STC1 structure with bound junction spacer (in orange) and the tip of the recipient L‐TIR (in gray). Labeled residue side chains shown as sticks belong to the cd B ‐binding site. Diagram of observed interactions between TnpA and tip of donor R‐TIR, junction spacer and recipient L‐TIR in STC1. Interactions with the rest of R‐TIR are identical to those in the PCC. In vitro integration of TIR junction mimics with modified L‐TIRs or spacer sequence. Lane 1, pUC19 alone after 24 h of incubation in reaction buffer omitting only oligonucleotide substrate. White triangles mark the TIR ends. “M”, base pair marker. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Article Snippet: DNA integration assay in vitro and TSD identification Qualitative assays were conducted in a total volume of 100 μl with the same reaction buffer as in the circularization assay using 2 μM DNA oligonucleotides, 0.25 μM TnpA, and 200 ng of pUC19 (Invitrogen) as the random DNA target.

    Techniques: Binding Assay, Periodic Counter-current Chromatography, Labeling, In Vitro, Modification, Sequencing, Incubation, Marker, Plasmid Preparation

    Characterization of IS C th4 transposase strand transfer reactions Schematic of assay to detect formation of Figure‐eight intermediate. pUC19 was modified to include the 35‐bp L-TIR and R‐TIR sequences (pUC19LR) or L‐TIR only as a control (pUC19L). White triangles mark the TIR ends. Strand transfer of one TIR to the other (depicted as orange arrow) was detected using PCR primers det1 and det2 ( Appendix Table S1 ). Detection of strand transfer by PCR. pUC19L or pUC19LR was used as substrate in supercoiled (sc.) or linear (lin.) form. Activity as assessed by a PCR product of expected size was detected when pUC19LR was linearized (lane 2). Other reactions yielded only background products of incorrect sizes. (top) Sequences of four unique Figure‐eight junction intermediates, obtained by cloning the PCR band in lane 2 of panel B, and screening 24 colonies, eight of which corresponded to junctions. Presented sequences are the reverse complements of those detected. (bottom) For reference, the 8‐bp sequences adjacent to each cloned TIR in pUC19LR are underlined. Schematic of assay to detect single‐end (rlx) and double‐end (lin) joined products. The target plasmid is supercoiled pUC19 (sc), and the reaction shown depicts junction integration. TIR sequence is in blue, junction spacer sequence in orange. Yellow dots indicate the 3′‐OH groups. In vitro integration of R‐TIR as a function of TIR length. Lane 1, pUC19 alone at t = 0. Lane 2, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. White triangle marks the TIR end. Comparison of in vitro integration of a random 26‐mer (lanes 3–5) and an oligonucleotide where 14 bp of the R‐TIR were replaced by random sequence (in green); bp 13–26 of the TIR are unchanged (lanes 6–8). For both substrates, the low level of relaxed plasmid formation after 24 h is similar to that of pUC19 alone after 20 h of incubation in reaction buffer when only the TIR oligonucleotide is omitted (lane 2). White triangle marks the TIR end. In vitro integration of transposon junction mimic as a function of junction length (depicted in orange). Green indicates 9 bp of random DNA added to oligonucleotide ends to direct correct annealing. The color scheme is maintained throughout. Lane 1, pUC19 alone at t = 0. White triangles mark the TIR ends. Comparison of in vitro integration of L‐TIR26 (lanes 3–8), R‐TIR flanked by 41 bp of random DNA (R35r41, lanes 9–14), and TIR junction mimic with 6‐bp T/A‐rich spacer (R35(j6)L35, lanes 15–20). For each reaction shown, time points are as follows: 0, 0.5, 1, 2, 5, and 24 h. Lane 2, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. (top) Ethidium bromide‐stained gel. (bottom) Same gel visualized by fluorescence detection. The yellow square indicates the location of 5′‐FAM. White triangles mark the TIR ends. (top) Identified target sites in pUC19 with the central 8 bp in bold corresponding to the sequenced linear products from the reaction in panel G (lane 17). (bottom) Target sites from Clostridium thermocellum (ATCC 24705) show a similar preference for A/T-rich sequences. Data information: “M”, base pair marker with indicated size of standards. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Structures of ISCth4 transpososomes reveal the role of asymmetry in copy‐out/paste‐in DNA transposition

    doi: 10.15252/embj.2020105666

    Figure Lengend Snippet: Characterization of IS C th4 transposase strand transfer reactions Schematic of assay to detect formation of Figure‐eight intermediate. pUC19 was modified to include the 35‐bp L-TIR and R‐TIR sequences (pUC19LR) or L‐TIR only as a control (pUC19L). White triangles mark the TIR ends. Strand transfer of one TIR to the other (depicted as orange arrow) was detected using PCR primers det1 and det2 ( Appendix Table S1 ). Detection of strand transfer by PCR. pUC19L or pUC19LR was used as substrate in supercoiled (sc.) or linear (lin.) form. Activity as assessed by a PCR product of expected size was detected when pUC19LR was linearized (lane 2). Other reactions yielded only background products of incorrect sizes. (top) Sequences of four unique Figure‐eight junction intermediates, obtained by cloning the PCR band in lane 2 of panel B, and screening 24 colonies, eight of which corresponded to junctions. Presented sequences are the reverse complements of those detected. (bottom) For reference, the 8‐bp sequences adjacent to each cloned TIR in pUC19LR are underlined. Schematic of assay to detect single‐end (rlx) and double‐end (lin) joined products. The target plasmid is supercoiled pUC19 (sc), and the reaction shown depicts junction integration. TIR sequence is in blue, junction spacer sequence in orange. Yellow dots indicate the 3′‐OH groups. In vitro integration of R‐TIR as a function of TIR length. Lane 1, pUC19 alone at t = 0. Lane 2, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. White triangle marks the TIR end. Comparison of in vitro integration of a random 26‐mer (lanes 3–5) and an oligonucleotide where 14 bp of the R‐TIR were replaced by random sequence (in green); bp 13–26 of the TIR are unchanged (lanes 6–8). For both substrates, the low level of relaxed plasmid formation after 24 h is similar to that of pUC19 alone after 20 h of incubation in reaction buffer when only the TIR oligonucleotide is omitted (lane 2). White triangle marks the TIR end. In vitro integration of transposon junction mimic as a function of junction length (depicted in orange). Green indicates 9 bp of random DNA added to oligonucleotide ends to direct correct annealing. The color scheme is maintained throughout. Lane 1, pUC19 alone at t = 0. White triangles mark the TIR ends. Comparison of in vitro integration of L‐TIR26 (lanes 3–8), R‐TIR flanked by 41 bp of random DNA (R35r41, lanes 9–14), and TIR junction mimic with 6‐bp T/A‐rich spacer (R35(j6)L35, lanes 15–20). For each reaction shown, time points are as follows: 0, 0.5, 1, 2, 5, and 24 h. Lane 2, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. (top) Ethidium bromide‐stained gel. (bottom) Same gel visualized by fluorescence detection. The yellow square indicates the location of 5′‐FAM. White triangles mark the TIR ends. (top) Identified target sites in pUC19 with the central 8 bp in bold corresponding to the sequenced linear products from the reaction in panel G (lane 17). (bottom) Target sites from Clostridium thermocellum (ATCC 24705) show a similar preference for A/T-rich sequences. Data information: “M”, base pair marker with indicated size of standards. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Article Snippet: DNA integration assay in vitro and TSD identification Qualitative assays were conducted in a total volume of 100 μl with the same reaction buffer as in the circularization assay using 2 μM DNA oligonucleotides, 0.25 μM TnpA, and 200 ng of pUC19 (Invitrogen) as the random DNA target.

    Techniques: Modification, Polymerase Chain Reaction, Activity Assay, Clone Assay, Plasmid Preparation, Sequencing, In Vitro, Incubation, Staining, Fluorescence, Marker

    Characterization of TIR junction integration and sedimentation analysis of DNA :TnpA complexes In vitro integration of TIR junction mimics. Modifications to R35(j6)L35 are as shown schematically on the right. The 5′‐end of the L‐TIR was labeled with Cy5 (blue triangle) and the 5′‐end of the R‐TIR with FAM (yellow square). The DNA color code corresponds to that in Fig 2 . Lane 2, pUC19 alone at t = 0. Lane 3, pUC19 after incubation for 24 h with non‐related 26‐mer with one 5′‐OH FAM label. White triangles mark the TIR ends. In vitro integration of oligonucleotide junctions using a linearized pUC19 target. Lane 1, supercoiled pUC19 alone at t = 0. Lane 3, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. Sedimentation velocity analytical ultracentrifugation (SV AUC) analysis of the complex formed between TnpA and junction oligonucleotide R35(j6)L35 as a function of increasing DNA concentration. SV AUC analysis of the complex formed between TnpA and the 35‐bp R-TIR flanked by 41 bp of random sequence, R35r41, as a function of increasing DNA concentration. The difference in s ‐values between the two complexes (7.9 vs. 8.5 S) may reflect differences in the stability of the complexes with different DNAs in the timescale of the experiment. Data information: “M”, base pair marker with indicated size of standards. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Structures of ISCth4 transpososomes reveal the role of asymmetry in copy‐out/paste‐in DNA transposition

    doi: 10.15252/embj.2020105666

    Figure Lengend Snippet: Characterization of TIR junction integration and sedimentation analysis of DNA :TnpA complexes In vitro integration of TIR junction mimics. Modifications to R35(j6)L35 are as shown schematically on the right. The 5′‐end of the L‐TIR was labeled with Cy5 (blue triangle) and the 5′‐end of the R‐TIR with FAM (yellow square). The DNA color code corresponds to that in Fig 2 . Lane 2, pUC19 alone at t = 0. Lane 3, pUC19 after incubation for 24 h with non‐related 26‐mer with one 5′‐OH FAM label. White triangles mark the TIR ends. In vitro integration of oligonucleotide junctions using a linearized pUC19 target. Lane 1, supercoiled pUC19 alone at t = 0. Lane 3, pUC19 after incubation for 24 h in reaction buffer omitting only the TIR oligonucleotide. Sedimentation velocity analytical ultracentrifugation (SV AUC) analysis of the complex formed between TnpA and junction oligonucleotide R35(j6)L35 as a function of increasing DNA concentration. SV AUC analysis of the complex formed between TnpA and the 35‐bp R-TIR flanked by 41 bp of random sequence, R35r41, as a function of increasing DNA concentration. The difference in s ‐values between the two complexes (7.9 vs. 8.5 S) may reflect differences in the stability of the complexes with different DNAs in the timescale of the experiment. Data information: “M”, base pair marker with indicated size of standards. Labels rlx, lin, and sc mark positions of relaxed, linear, and supercoiled plasmid forms, respectively. Source data are available online for this figure.

    Article Snippet: DNA integration assay in vitro and TSD identification Qualitative assays were conducted in a total volume of 100 μl with the same reaction buffer as in the circularization assay using 2 μM DNA oligonucleotides, 0.25 μM TnpA, and 200 ng of pUC19 (Invitrogen) as the random DNA target.

    Techniques: Sedimentation, In Vitro, Labeling, Incubation, Concentration Assay, Sequencing, Marker, Plasmid Preparation

    The SMR domain of SOT1 has nuclease activity. ( A ) Purified MBP, SOT1 SMR , and Gm-SOT1 SMR used for nuclease activity were resolved by SDS/PAGE with Coomassie Brilliant Blue (CBB) staining or with the analyses of immunoblot using the MBP antibody. The marker sizes are shown to the Left . ( B ) DNA endonuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR at different concentrations were incubated with 5 ng pUC19 plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . The reactions were stopped by loading buffer and were electrophoresed on 1.2% (wt/vol) agarose gels. Parallel experiments were carried out with EcoRI to linearize pUC19 or with H 2 O as a control. OC, open circular; CCC, covalently closed-circular forms of pUC19. ( C ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on DNA nicking activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 5 ng pUC19 was incubated with H 2 O, MgCl 2 , CaCl 2 , or MnCl 2 and 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR . The concentration of each cation was 3 mM. ( D ) Analyses of DNA nicking activity of Gm-SOT1. MBP and Gm-SOT1 were incubated with 5 ng pUC19 plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . ( E ) RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( F ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR were incubated with total wild-type Arabidopsis RNA in the presence of MgCl 2 , CaCl 2 , or MnCl 2 . The concentration of each cation was 3 mM. ( G ) Accumulation of total RNAs in WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 plants. A total of 3 μg total RNAs from 12-d-old WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 seedlings were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( H ) Analyses of RNA nuclease activity of Gm-SOT1. MBP and Gm-SOT1 with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were detected by electrophoretic separation in agarose/formaldehyde gels with ethidium bromide staining.

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

    Article Title: PPR-SMR protein SOT1 has RNA endonuclease activity

    doi: 10.1073/pnas.1612460114

    Figure Lengend Snippet: The SMR domain of SOT1 has nuclease activity. ( A ) Purified MBP, SOT1 SMR , and Gm-SOT1 SMR used for nuclease activity were resolved by SDS/PAGE with Coomassie Brilliant Blue (CBB) staining or with the analyses of immunoblot using the MBP antibody. The marker sizes are shown to the Left . ( B ) DNA endonuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR at different concentrations were incubated with 5 ng pUC19 plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . The reactions were stopped by loading buffer and were electrophoresed on 1.2% (wt/vol) agarose gels. Parallel experiments were carried out with EcoRI to linearize pUC19 or with H 2 O as a control. OC, open circular; CCC, covalently closed-circular forms of pUC19. ( C ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on DNA nicking activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 5 ng pUC19 was incubated with H 2 O, MgCl 2 , CaCl 2 , or MnCl 2 and 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR . The concentration of each cation was 3 mM. ( D ) Analyses of DNA nicking activity of Gm-SOT1. MBP and Gm-SOT1 were incubated with 5 ng pUC19 plasmid DNA at 25 °C for 60 min in the presence of 3 mM MgCl 2 . ( E ) RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . MBP, SOT1 SMR , and Gm-SOT1 SMR with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( F ) Effects of Mg 2+ , Ca 2+ , and Mn 2+ on RNA nuclease activities of the SMR domains of SOT1 and Gm-SOT1 SMR . A total of 100 nM MBP, SOT1 SMR , and Gm-SOT1 SMR were incubated with total wild-type Arabidopsis RNA in the presence of MgCl 2 , CaCl 2 , or MnCl 2 . The concentration of each cation was 3 mM. ( G ) Accumulation of total RNAs in WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 plants. A total of 3 μg total RNAs from 12-d-old WT, sot1-3/35S:SOT1 SMR -HA , and sot1-3 seedlings were separated in agarose/formaldehyde gels and observed by ethidium bromide staining. ( H ) Analyses of RNA nuclease activity of Gm-SOT1. MBP and Gm-SOT1 with different concentrations were incubated with total wild-type Arabidopsis RNA at 25 °C for 30 min. The reaction products were detected by electrophoretic separation in agarose/formaldehyde gels with ethidium bromide staining.

    Article Snippet: The pUC19 (Takara) plasmid DNA (5 ng μL−1 ) was incubated with MBP, SOT1SMR , SOT1SMR variants, Gm-SOT1SMR , Gm-SOT1SMR variants, or Gm-SOT1 in buffer [20 mM phosphate, pH 7.5, 160 mM NaCl, 40 mM KCl, and 4% (vol/vol) glycerol] at 25 °C for 60 min.

    Techniques: Activity Assay, Purification, SDS Page, Staining, Marker, Incubation, Plasmid Preparation, Countercurrent Chromatography, Concentration Assay