The GenBuilder™ DNA Assembly kit has shown the highest cloning efficiency among
competitor kits with 5 fragment cloning
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.
Reduced Reaction Incubation Time
GenBuilder™ DNA Assembly has shown optimized results with a reduced reaction time of 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
Competitors A and B
GenBuilder™ Plus vs. DNA Assembly kits from Competitors A and B
Graph A. Impact of the overlapping base pair length
with cloning of 5 DNA inserts into one vector. Various PCR fragments
with overlapping 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.
Graph B. Impact of DNA fragment concentration with
cloning of 5 DNA inserts into one vector. Cloning of 5 DNA inserts of
various 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.
Graph C. Performance of Assembly kits with the
increase of the amount of DNA fragments. Multiple fragment cloning
impact with various DNA Assembly methods. 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.
Figure 4 (graphs A, B and C). Comparison study of GenBuilder™ Plus DNA Assembly
kit with Competitors A and B.
Multiple Fragment Clones Assembly Success with GenBuilder™ and GenBuilder™ Plus
Figure 5: Varying number of DNA fragment assemblies using GenBuilder™ and GenBuilder™
Plus kits.
Cloning reactions with 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 shown significant
cloning efficiency.
