DAPE cloning with modified primers for producing designated lengths of 3’ single-stranded ends in PCR products
Fig 5
(A) A schematic diagram of gRNA cloning into the px330-puro vector. After linearization with BbsI treatment, 50 ng of the linear vector was subjected to cloning procedures with 1 μl (5%) of 54 bp PCR products for DAPE or the same amount of 52 bp PCR products for Golden Gate cloning. While the linearized vector was purified using purification kits, the crude PCR products were directly used for cloning. (B) Comparison of Golden Gate cloning and DAPE. Although Golden Gate cloning (left) yielded more colonies than DAPE (right), the former also resulted in a higher number of non-positive clones. DAPE without PTs failed to form positive clones. A linear vector without gRNA was used as a negative control (labeled as "linear vector"). Single asterisk; P-values lower than 0.05, double asterisk; lower than 0.01. The triple asterisks represent P value lower than 0.001, derived from triple experiments. (C) Representative individual images of triplet (B). (D) Cloning accuracies of Golden Gate cloning and DAPE were validated by sequencing. Five randomly selected clones were sequenced to identify the orientation and sequences of the gRNA. Only DAPE with 5 PTs showed 100% accuracy of cloning. CFU; colony forming unit.