Competitive performance of the GenBuilder™ cloning kit
Highest cloning efficiency with five fragments cloning
GenBuilder™ DNA Assembly has the highest cloning efficiency when cloning five gene fragments into one vector
Figure 1. Cloning five PCR fragments into a vector using various DNA Assembly methods. Five PCR fragments from 0.7 kb to 1.6 kb in length were assembled into pUC57 in a single-step assembly reaction. The number of colonies recovered from 1/10 of the cloning reaction were counted for each reaction. Error bars represent standard deviations of at least three independent experiments. GenBuilder™ DNA Assembly had the best assembly performance among all competing methods.
Save time on incubation of the DNA Assembly reaction
GenBuilder™ DNA Assembly achieves maximum results in just 15 minutes
Figure 2. Comparing the effect of time of incubation with GenBuilder™ master-mix on cloning efficiency. Two separate reactions were performed for DNA Assembly into the pUC57 vector - one using a single 1 kb PCR purified fragment, and another using two 1.5 kb PCR purified fragments. GenBuilder™ DNA assembly reactions were performed at 50°C for either 15 minutes or 60 minutes. Following the DNA assembly reaction, 1 µl assembly product was transformed into chemically competent DH10B cells. After recovery in 1 mL SOC medium, 100 µl of the cells were spread on selective plates. Using both one fragment (panel 1) and two fragments (panel 2), no detectable difference was observed between the number of colonies recovered when incubated with the GenBuilder™ master-mix at 50°C for 50 minutes (blue bars) or 15 minutes (yellow bars). All colonies were >90% PCR positive.
Save time on PCR clean-up
Figure 3. Cloning two unpurified PCR fragments into a vector using various DNA Assembly methods. Two fragments were used for DNA assemble directly after PCR, without purification or fragment enrichment. The PCR fragments were assembled into pUC57 in a single-step assembly reaction. The number of colonies recovered from 1/10th of the cloning reaction were counted per reaction. Error bars represent standard deviations of at least three independent experiments. GenBuilder™ DNA Assembly had among the best performances using unpurified PCR products.
Comparison of GenBuilder™ Plus DNA Assembly kit with Gibson Assembly
GenBuilder™ Plus vs. Gibson Assembly
Figure 4. Comparison of GenBuilder™ with Gibson Assembly. A. Cloning 5 inserts into one vector with various lengths of DNA overlap using various DNA Assembly methods. Various PCR fragments with overlap lengths of 10 bp, 15 bp, 25 bp, 30 bp, and 40 bp were assembled into pUC57 in a single-step assembly reaction. The number of colonies recovered from 1/10th of the cloning reaction were counted per reaction. B. Cloning 5 inserts of various DNA concentrations into one vector using various DNA Assembly methods. Various concentrations of five PCR fragments (0.01 pmol, 0.02 pmol, 0.05 pmol, 0.1 pmol, and 0.2 pmol) were assembled into pUC57 in a single-step assembly reaction. The number of colonies recovered from 1/10th of the cloning reaction were counted per reaction. C. Multiple fragment cloning using various DNA Assembly methods. In various assembly reactions, 6, 8, 10 and 12 PCR fragments were assembled into linearized pUC57 vector in a single-step assembly reaction. The number of colonies recovered from 1/10th of the cloning reaction were counted for each reaction. GenBuilder™ Plus DNA Assembly had the best assembly performance.
Twelve fragments cloning with GenBuilder™ vs. GenBuilder™ Plus
Figure 5. Multiple (12) fragment cloning using GenBuilder™ and GenBuilder™ Plus. In various assembly reactions, 6, 8, 10 and 12 PCR fragments were assembled into linearized pUC57 vector in a single-step assembly reaction. The number of colonies recovered from 1/10th of the cloning reaction were counted for each reaction. GenBuilder™ Plus has significantly better cloning efficiency.
