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Found 4 matching solutions for this experiment
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The DNA assembly was carried out in 10 μl reaction comprised of ~50 ng Acceptor Vector and twice as many molar of the insert parts, in addition to 1 μl 1 mg/ml BSA (NEB), 1 μl T4 DNA ligase buffer (NEB or Thermo Fisher Scientific), 0.5 μl AarI (Thermo Fisher Scientific) for cloning in mUAV and Level 2 Acceptor Vectors or Eco31I (BsaI) (Thermo Fisher Scientific) for cloning in Level 1 Acceptor Vectors, and 0.5 μl T4 DNA ligase (NEB or Thermo Fisher Scientific). For reactions with AarI, extra 0.2 μl 50x oligos (0.025 mM) of the enzyme recognition sites were added. The one-tube reaction was incubated in a thermocycler for five times cycles of (37°C for 5 min, 16°C for 10 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. For the assembly of the 16 TU construct, the reaction was set in 20 μl with double amount of buffers and enzymes and the thermocycling conditions were altered to: 40 cycles of (37°C for 2.5 min, 16°C for 5 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. The sequences of all the primers and the resulting plasmids have been deposited to the public database Edinburgh Datashare (http://dx.doi.org/10.7488/ds/2261). |
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| Protocol tips |
| The DNA assembly was carried out in 10 μl reaction comprised of ~50 ng Acceptor Vector and twice as many molar of the insert parts, in addition to 1 μl 1 mg/ml BSA (NEB), 1 μl T4 DNA ligase buffer (NEB or Thermo Fisher Scientific), 0.5 μl AarI (Thermo Fisher Scientific) for cloning in mUAV and Level 2 Acceptor Vectors or Eco31I (BsaI) (Thermo Fisher Scientific) for cloning in Level 1 Acceptor Vectors, and 0.5 μl T4 DNA ligase (NEB or Thermo Fisher Scientific). For reactions with AarI, extra 0.2 μl 50x oligos (0.025 mM) of the enzyme recognition sites were added. The one-tube reaction was incubated in a thermocycler for five times cycles of (37°C for 5 min, 16°C for 10 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. For the assembly of the 16 TU construct, the reaction was set in 20 μl with double amount of buffers and enzymes and the thermocycling conditions were altered to: 40 cycles of (37°C for 2.5 min, 16°C for 5 min) followed by 5 min digestion at 37°C and 5 min deactivation at 80°C. The sequences of all the primers and the resulting plasmids have been deposited to the public database Edinburgh Datashare (http://dx.doi.org/10.7488/ds/2261). |
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For proof of principle modular assembly of ∼1 kb DNA from 4 fragments by MetClo, assembly reactions were set up using 60 fmol of each donor plasmid prepared from normal DH10B cells, 60 fmol of assembly vector prepared from DH10B strains stably expressing compatible methylase, 1,000 U T4 DNA ligase (NEB), and 5 U BsaI (NEB) or BpiI (Thermo Fisher Scientific) or 2.5 U LguI (Thermo Fisher Scientific) in 20 μl 1× T4 ligase buffer (NEB). The reaction condition was: 37°C 15 min, followed by 45 cycles of 37°C 2 min plus 16°C 5 min, then 37°C 20 min, and 80°C 5 min. Assembly reactions were transformed into normal DH10B chemical competent cells, and plated on LB plates with AIX selection (ampicillin 100 μg/ml, IPTG 100 μM, X-gal 50 μg/ml) at 37°C overnight. White colonies were expanded and screened by restriction digestion using the corresponding restriction enzyme and by DNA sequencing. |
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| Protocol tips |
| For proof of principle modular assembly of ∼1 kb DNA from 4 fragments by MetClo, assembly reactions were set up using 60 fmol of each donor plasmid prepared from normal DH10B cells, 60 fmol of assembly vector prepared from DH10B strains stably expressing compatible methylase, 1,000 U T4 DNA ligase (NEB), and 5 U BsaI (NEB) or BpiI (Thermo Fisher Scientific) or 2.5 U LguI (Thermo Fisher Scientific) in 20 μl 1× T4 ligase buffer (NEB). The reaction condition was: 37°C 15 min, followed by 45 cycles of 37°C 2 min plus 16°C 5 min, then 37°C 20 min, and 80°C 5 min. Assembly reactions were transformed into normal DH10B chemical competent cells, and plated on LB plates with AIX selection (ampicillin 100 μg/ml, IPTG 100 μM, X-gal 50 μg/ml) at 37°C overnight. White colonies were expanded and screened by restriction digestion using the corresponding restriction enzyme and by DNA sequencing. |
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All Golden Gate reactions were performed in a total volume of 15 µl. The final reaction volume contained 1-fold concentrated T4 ligase buffer (Promega, Madison, US). Prepared reaction mixtures (ligase buffer, acceptor plasmid, insert(s)) was adjusted to 13.5 µl with ddH2O. In a final step, the corresponding enzymes were quickly added. First, a volume of 0.5 µl of the respective restriction enzyme BbsI (5 units; ThermoFisherScientific, Waltham, US) or BsaI-HF®v2 (10 units; New England Biolabs, Ipswich, US) and then 1 µl (1–3 units) of T4 ligase (Promega, Madison, US) was added. Golden Gate reactions were carried out by default under following conditions: a) Enzymatic restriction 37 °C (2 min) [40 passes]; b) Ligation 20 °C (5 min) [40 passes] and c) enzyme inactivation: 80 °C (20 min). |
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| Protocol tips |
| All Golden Gate reactions were performed in a total volume of 15 µl. The final reaction volume contained 1-fold concentrated T4 ligase buffer (Promega, Madison, US). Prepared reaction mixtures (ligase buffer, acceptor plasmid, insert(s)) was adjusted to 13.5 µl with ddH2O. In a final step, the corresponding enzymes were quickly added. First, a volume of 0.5 µl of the respective restriction enzyme BbsI (5 units; ThermoFisherScientific, Waltham, US) or BsaI-HF®v2 (10 units; New England Biolabs, Ipswich, US) and then 1 µl (1–3 units) of T4 ligase (Promega, Madison, US) was added. Golden Gate reactions were carried out by default under following conditions: a) Enzymatic restriction 37 °C (2 min) [40 passes]; b) Ligation 20 °C (5 min) [40 passes] and c) enzyme inactivation: 80 °C (20 min). |
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Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O. It is crucial for the reactions that the entry vectors are purified and diluted in pure water since buffer components might inhibit the Golden gate assembly. All reactions were incubated using the following cycling conditions: 2 min at 37 °C, 5 min at 20°C for 50 cycles, then 5 min at 50°C and 5 min at 80°C. 10µl of the reaction mix were used to transform chemical competent cells (Top10 strain from Life Technologies). |
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| Protocol tips |
| Reactions were set up using 20 fmol of each Golden Gate entry vector and the destination vector, 10 U of BsaI Restriction Enzyme (Thermo, Fisher Fast Digest Enzyme Eco31I) and 30 U of T4 DNA Ligase (Thermo, Fisher) in 2 µl 10x ligation buffer (10x ligation buffer was prepared by supplementing the Thermo, Fisher FastDigest buffer with 10mM dATP and 100mM DTT) to a final reaction volume of 20 µl with ddH2O. It is crucial for the reactions that the entry vectors are purified and diluted in pure water since buffer components might inhibit the Golden gate assembly. All reactions were incubated using the following cycling conditions: 2 min at 37 °C, 5 min at 20°C for 50 cycles, then 5 min at 50°C and 5 min at 80°C. 10µl of the reaction mix were used to transform chemical competent cells (Top10 strain from Life Technologies). |
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