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. 2020 Jul 29;5(31):19702–19714. doi: 10.1021/acsomega.0c02396

Figure 2.

Figure 2

Constructing custom TALENs with a new TALEN protocol. (A) TALEN plasmid-transformed E. coli colonies grew on an agar plate. There were few blue colonies in all plates. (B) Colony PCR detection of randomly picked white colonies. Eight colonies were detected for two RELA TALENs, and four colonies were detected for two TALENs of RELB, CREL, NFKB1, and NFKB2. The full-length PCR products are 2051 bp long. The ladder effect indicates a successful assembly. All colony PCR-detected colonies were cultivated to extract plasmids. The obtained plasmids were digested with EcoRI in which the positive colonies produced a full-length 3537-bp TALEN fragment, but the negative colonies (without inserted sequence) produced a 2143-bp band. (C) PCR and EcoRI detections of TALENs prepared with (up) or without (down) colony screening. (D) Two colonies were screened for each CRISPR/Cas9-sgRNA. M1: DL2000 DNA marker (Code No.3427A; Takara); M2: DL10000 DNA marker (code no. 3584A; Takara); and M3: 100-bp DNA ladder (code no. 3422A; Takara).