Table 1. . Optimization of overlap extension PCR cloning parameters.

Experiment 1
Polymerase	Touchdown	PCR cleanup	Positive/total screened clones (PCR)	Transformants
Mixed (Taq and Phusion)	Yes	No	10/10	309
Mixed (Taq and Phusion)	No	No	10/10	51
Phusion only	Yes	No	0	0
Phusion only	No	No	0	0

Experiment 2
Polymerase	Touchdown	PCR cleanup	Positive/total screened clones (PCR)	Transformants
Mixed (Taq and Phusion)	Yes	Yes	10/10	98
Mixed (Taq and Phusion)	Yes	No	10/10	28

Experiment 3
Polymerase	Touchdown	PCR cleanup	Positive/total screened clones (PCR)	Positive/total screened clones (restriction digest)
Mixed (Taq and Phusion)	Yes	No	13/13	3/13

Experiment 1 compares effects of different polymerases and touchdown conditions in PCR-2 on cloning efficiency. Experiment 2 tests effect of PCR purification of PCR-1 product. Experiment 3 tests efficacy of modified PCR extension cloning technique when the same antibiotic resistance was used in source and target plasmids. Positive/total screened clones (PCR) is the number of samples tested for the FOP gene using PCR. Positive/total screened clones (restriction digest) is the number of samples tested with restriction digestion for presence of FOP/pGATE-1 vector vs FOP/pDONR207 vector.