DNA Ligation Kit, Mighty Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer	The ligation reaction mixture should not be used directly in electroporation. Instead, ligated DNA should be ethanol precipitated and dissolved in a low salt buffer, such as TE buffer, prior to use for electroporation.

Upstream tips
Always use the supplied buffer to avoid high salt concentration or add ATP to a compatible buffer
Protocol tips
The ligation reaction mixture should not be used directly in electroporation. Instead, ligated DNA should be ethanol precipitated and dissolved in a low salt buffer, such as TE buffer, prior to use for electroporation.

Publication protocol

The DNA fragment of the onion RanGAP (1–497 amino-acid residues) was amplified by PCR using a PrimeStar DNA polymerase and a primer set ACRanGAP1_3 (Fig. S1A) to add the restriction enzyme sites KpnI and BamHI at the 5′- and 3′-end, respectively and was inserted in a pBluescriptII SK(−). The DNA fragment was then isolated from this plasmid using KpnI and BamHI restriction enzymes, and ligated into an expression vector pET-32b (Novagen, EMD Bioscience, Inc.) cut by KpnI and BamHI using DNA Ligation Kit, Mighty Mix (Takara Bio Inc.). The obtained plasmid was transformed into Escherichia coli BL21 (DE3) strain. The expressed polypeptides that were insoluble in the E. coli cells were solubilized with urea and then purified and dialyzed to remove urea using a His-Bind purification kit (Novagen, EMD Bioscience, Inc.) according to the manufacturer's instructions. Purified polypeptide (400 μg) mixed with a Freund's Complete Adjuvant (FCA, Cosmo Bio Co.) was injected into a rabbit (New Zealand white). After 2 weeks and 4 weeks, the rabbit was inoculated with the polypeptide (200 μg) mixed with Freund's Incomplete Adjuvant (FIA, Cosmo Bio Co.). Two weeks later, we prepared a serum from the blood collected from the immunized rabbit. To obtain an antibody immunoreactive to the onion RanGAP, the purified polypeptide (5 μg) was separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane. After the transfer, the membrane was immersed in a solution of 5% skim milk and TBS, consisting of 50 mM Tris-HCl (pH 7.5) and 0.15 M NaCl and a part (about 1 cm wide) of the membrane corresponding to the size of the polypeptide was cut out with clean scissors. The strip of the membrane was reacted with 5-fold dilution of the serum obtained from the immunized rabbit in a phosphate-buffered saline (PBS; 1.5 mM KH2PO4, 137 mM NaCl, 10 mM Na2HPO4, pH 7.4) with 0.05% Tween 20 at room temperature for 1 h. After washing the strip with PBS in 0.05% Tween 20 four times, the stripe was incubated in 0.1 M glycine-HCl pH 2.5 at room temperature for 7 min to elute the antibody specific to the onion RanGAP. The eluate was collected and neutralized with ice-chilled 1 M Tris. Finally, NaN3 was added to the eluate and the eluate was stored at 4°C. For immunostaining, we used mouse monoclonal anti ß-tubulin (Sigma-Aldlich Co.), sheep polyclonal anti-tubulin (Cytoskeleton, Inc.), mouse monoclonal antibody against the 65 kDa MAP53 (renamed as anti-BY2MAP65) and the anti-onion RanGAP antibody as described above. To prepare onion crude protein, root tips of 4-day-old seedlings were crushed in a liquid nitrogen with a mortar and pestle and homogenized in a Protein Extraction Buffer (GE Healthcare). Then the homogenate was centrifuged at 20,000 × g for 30 s at 4°C and the obtained supernatant was collected as the onion crude protein. The onion crude protein was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane, which was then incubated in 5% skim milk in TBS. Immunoblotting analyses were performed with the primary antibodies described above, and immuno-reactive bands were visualized with a peroxidase labeled goat anti-rabbit IgG (H + L) (Kirkegaard & Perry Laboratories) or VECTASTAIN Universal Elite ABC Kit (Vector Laboratories Ltd.) using a Pierce Western Blotting substrate (Thermo Fisher Scientific, Inc.). Negative control experiments were carried out using a preimmune serum or a mixture of purified antibody with antigen that was preincubated overnight at 4°C.

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