Fig. 4

Increasing Deletions Using Gibson reaction. a pUC19 plasmid was cut with KpnI and this restriction site was substituted with an AflII site using Gibson Deletion. Single stranded oligos were used as AflII site donor DNA for Gibson reaction. The oligonucleotides used were designed to delete 0, 10 (5 on each side), 20 (10 on each side), 30 (15 on each side), 40 (20 on each side), 50 (25 on each side), 60 (30 on each side), 80 (40 on each side), 100 (50 on each side), and 200 (100 on each side) nucleotides flanking the KpnI/AflII sites. The red and yellow lines and boxes represent homologous sequences of 20 nt on the single stranded oligonucleotides (yellow and red lines) and on the vector (yellow and red boxes). b-c After Gibson reaction, each sample was transformed and 1/10 or 9/10 of the transformed bacteria were plated on LB plates supplemented with carbenicillin. The table reports the number of growing colonies for each reaction also represented in the bar graph in c. d-e Number of correct clones after diagnostic cut with KpnI and AflII (d) or after Sanger sequence of a subset of samples (e)