HindIII-HF®

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	The plasmid DNA of pBS3ndd was isolated using the alkaline lysis method, then linearized by digestion with EcoRI-HF and HindIII-HF for 3 h. Cut vector was precipitated with two volumes of absolute ethanol, incubated at −80 °C for 1 h, and then harvested by centrifugation at 16,000×g at room temperature for 10 min. The DNA pellet was rinsed once with 70% ethanol, air-dried, and resuspended with TE buffer.

Protocol tips

The plasmid DNA of pBS3ndd was isolated using the alkaline lysis method, then linearized by digestion with EcoRI-HF and HindIII-HF for 3 h. Cut vector was precipitated with two volumes of absolute ethanol, incubated at −80 °C for 1 h, and then harvested by centrifugation at 16,000×g at room temperature for 10 min. The DNA pellet was rinsed once with 70% ethanol, air-dried, and resuspended with TE buffer.

Publication protocol

The forward and reverse primers used to amplify four genomic regions, 18S, ITS, rbcL, and trnLF, were 5′-flanked with the sequence of pBluescript KS and pBluescript SK primers to serve as homology arms. All PCR insert fragments were generated using Q5 High-Fidelity DNA Polymerase. The plasmid DNA of pBS3ndd was isolated using the alkaline lysis method, then linearized by digestion with EcoRI-HF and HindIII-HF for 3 h. Cut vector was precipitated with two volumes of absolute ethanol, incubated at −80 °C for 1 h, and then harvested by centrifugation at 16,000×g at room temperature for 10 min. The DNA pellet was rinsed once with 70% ethanol, air-dried, and resuspended with TE buffer. For in vivo cloning, 5 µl of crude PCR product and 5 µl of 500 ng/µl cut vector were co-transformed into the competent cells, and the entire volume of transformation product was plated on LB agar containing kanamycin and IPTG

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