Figure 4.

Constructing Custom TALEs with New Monomers and Pipeline

(A and B) E. coli colonies on solid agar that were transformed with HNF4α-TALE1-VPR (A) and E47-TALE1-VPR (B). (C and D) Colony PCR detection of four groups of randomly selected white spots. HNF4α-TALE1-VPR (C) and E47-TALE1-VPR (D) groups contained eight colonies. Full-length PCR products should be 2,051 bp long. However, PCR products were often less prominent, whereas the “ladder effect” represented a robust indicator of successful assembly. (E and F) Colony identification by EcoRI digestion. All colonies picked from HNF4α-TALE1-VPR (E) and E47-TALE1-VPR (F) were cultivated for extracting plasmid. The extracted plasmids were digested with EcoRI, which cut out the full-length TALE of 3,537 bp. The incorrectly assembled TALEs produced a 2,143-bp band in EcoRI digestion. It can be seen that the identification results of colony PCR were in agreement with those of EcoRI digestion.