| Cat. No. | Product Name | Quantity | Price | Order |
|---|---|---|---|---|
|
L00701
|
GenBuilder™ Cloning Kit |
|
$136.00 |
|
|
L00744
|
GenBuilder™ Plus Cloning Kit |
|
$157.00 |
|
#1 You choose your samples, GenBuilder™ DNA Assembly delivers the results
#2 Best on the market, GenBuilder™ DNA Assembly outcompetes all other DNA Assembly kits with performance
#3 GenBuilder™ DNA Assembly- a single solution for multiple cloning applications
Applications include:
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.
GenScript's GenBuilder™ DNA Assembly technology helps you easily perform modifications to your DNA. The applications range from expressing synthetic pathways in living cells to analyzing protein function using saturation mutagenesis.
Figure 1. Pathway assembly using GenBuilder™. The violacein biosynthetic pathway (8 kb) was assembled using the GenBuilder™ DNA Assembly. A. The pathway consists of five genes, which were assembled as six separate fragments into the pUC57 vector. B. The pathway was correctly assembled, and was functional in producing the purple violacein pigment in DH10B cells. PCR and sequencing analysis further confirmed the seamless DNA assembly.
Figure 2. Using ssDNA as a bridge to create saturation mutagenesis for directed evolution. The RFP gene consisting of two fragments was mutated with ssDNA fragments. A. Q66 was targeted for NKK saturation mutagenesis in between 426 bp and 661 bp RFP sub-fragments. B. Codons 194, 195, and 196 were targeted for NKK saturation mutagenesis in between 810 bp and 271 bp RFP sub-fragments. The DNA fragments were assembled into pUC57 vector. C. Both reactions produced hundreds of colonies in different colors, indicating the successful introduction of various mutations in RFP. 24 clones were randomly picked for sequencing of the 194-196 saturation mutagenesis sample and found >92% of codon diversity
| Features | GenBuilder™ Cloning Kit | GenBuilder™ Plus Cloning Kit | |
| 1 | Unique need fulfilled by the kit | Multi-gene cloning for up to 6 fragments | Multi gene cloning for up to 12 fragments |
| 2 | Cost per reaction | $7.90 | $9.80 |
| 3 | Number of fragments that can be cloned into a linearized vector | 6 | 12 |
| 4 | Cloning efficiency (insert was cloned into the vector) % | >90% | >90% |
| 5 | Plasmid library construction | Suitable | Recommended |
| 6 | Minimum time taken for cloning the maximum number of inserts in a vector | 15 min | 15 min |
| 7 | Cloning unpurified PCR product | Efficient | Highly efficient |
| 8 | Assembly with ss oligos (single stranded oligos) | No | Yes |
| 9 | Can be used for high throughput cloning | Yes | Yes |
| 10 | Seamless cloning | Yes | Yes |
| 11 | Positive control | Linearized pUC57 with two fragments to reconstitute RFP for visual determination of cloning efficiency | Linearized pUC57 with two fragments to reconstitute RFP for visual determination of cloning efficiency |
Cloning methods using GenBuilder™ Cloning kit or Plus Cloning Kit
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Introduction, mechanism of action and selection guide of GenScript's featured DNA cloning kits
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