Neb t4 dna ligase

Lyophilized DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA), resuspended in water, quantitated by UV absorbance at 260 nm using a Thermo Scientific Nanodrop 2000c Spectrophotometer, and stored at −20°C. All samples were stored or mixed using DNA Lo-bind tubes (# 022431021).
Nanotubes were annealed at either 3 or 1.8 μM tile concentration by mixing each tile strand at 3 or 1.8 μM (final concentration), in Tris-Acetate-EDTA (TAE) and 12.4 mM MgCl₂. Nanopure water was added to achieve the appropriate concentration of components. All nanotube designs, except those which were to be ligated, were annealed using an Eppendorf Mastercycler PCR machine by heating the sample to 90°C, and cooling it to 25°C over a 6 h period. Nanotubes which were to be ligated were annealed by heating the sample to 90°C, and cooling it to 25°C over a 54 h period.
Nanotubes were ligated at 1.2 μM using New England Biolabs (NEB) T4 DNA Ligase (# M0202S; 20 000 units) at 1.0 units/μl and NEB T4 DNA Ligase buffer (# B0202S, 10×, 10 mM MgCl₂, 50 mM Tris-HCl, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) at 1×. Nanotubes were incubated at room temperature for at least 2 days in ligation solution preceding experiments.

The full‐length coding DNA sequence (CDS) of GhBZR3 was amplified from total cDNA by PCR with the primers listed in Table S3. PCR products were digested with SmaI and BamHI and then ligated into the pBI121‐GFP vector using NEB T4 DNA ligase (NEB, https://international.neb.com). The pBI121‐GhBZR3‐GFP and pBI121‐GFP vectors were transformed to Agrobacterium tumefaciens leaf and were further transiently transformed into 2‐week‐old tobacco leaves. The GFP signal was detected using a fluorescence microscope after 2 days (TCS SP5; Leica Microsystems, https://www.leica‐microsystems.com).