Eco31I (BsaI) (10 U/µL)

Protocol tips

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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).

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).

Publication protocol

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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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Eco31I (BsaI) (10 U/µL) below.

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