Seamless DNA Assembly Ultra Kit
Based on 2 publication(s) in Google Scholar
MCE Seamless DNA Assembly Ultra Kit is a next-generation recombinant cloning kit that allows the recombination of single or multiple DNA fragments in a single reaction.
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Storage :
-20℃, 2 years
Avoid repeated freeze-thaw cycles
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Tel: 609-228-6898 E-mail: [email protected] [email protected]
Description & Advantages
Based on gene recombination, seamless cloning is a simple, efficient, and fast DNA cloning technology. A 15-20 bp sequence homologous to the end of the linearized vector is added to the 5’ end of the insert fragment primers. In the presence of enzymes, recombination can be easily achieved.
MCE Seamless DNA Assembly Ultra Kit is a next-generation recombinant cloning kit that allows the recombination of single or multiple DNA fragments in a single reaction.
Features of MCE Seamless DNA Assembly Ultra Kit
1. Fast: Single fragment recombination can be completed in as little as 5 min, with a positive rate of over 95%.
2. High Fidelity: Utilizes an upgraded high-fidelity Taq DNA Ligase, significantly increasing the success rate of seamless cloning compared to regular Taq DNA Ligase.
3. High Transformation Efficiency: The mix includes a transformation enhancer, greatly improving transformation efficiency.
4. Optimized Buffer: The optimized buffer components provide greater stability and significantly enhanced antioxidant performance.
Protocol
1. Linearization of the vector
Select suitable cloning sites and completely linearize the vector by restriction digestion or inverse PCR. A high-fidelity DNA polymerase is recommended for PCR linearization. Purify the desired PCR product before assembly if nonspecific bands are present.
2. Primer design for insert fragments
Add sequences homologous to the two ends of the linearized vector to the 5' ends of the forward and reverse primers. A homologous overlap of approximately 15-20 bp is recommended. For multi-fragment assembly, design homologous overlaps between every adjacent DNA fragment.
3. DNA assembly reaction
Prepare the reaction on ice:
| Component | 10 μL reaction |
|---|---|
| Seamless DNA Assembly Mix Ultra | 5 μL |
| Linearized vector | 50-200 ng |
| Insert fragment | 10-200 ng/fragment |
| ddH2O | To 10 μL |
Note: Adjust vector and insert amounts according to fragment length and molar ratio. For inserts shorter than 200 bp, a higher insert-to-vector molar ratio may be used. The volume of unpurified PCR product should not exceed 20% of the total reaction volume.
4. Assembly program
| Assembly | Temperature | Time |
|---|---|---|
| 1-2 insert fragments | 50°C | 15 min |
| 3-5 insert fragments | 50°C | 30 min |
| Vector backbone >10 kb or insert >4 kb | 50°C | 30-60 min |
Immediately place the reaction tube on ice after incubation.
5. Transformation
Add 2-5 μL assembly product to 50 μL high-efficiency chemically competent cells. The assembly product should not exceed 10% of the competent-cell volume. Recover the cells, plate on the appropriate antibiotic-containing agar plate, and incubate inverted at 37°C overnight.
6. Positive clone identification
Screen single colonies by colony PCR, or culture selected colonies and confirm the recombinant plasmid by restriction analysis or sequencing.
Publications
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Journal Impact Factor
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Most Recent
Storage
-20℃, 2 years
Avoid repeated freeze-thaw cycles
Components
| Components | HY-K1041A-50 rxns |
|---|---|
| Seamless DNA Assembly Mix Ultra | 250 μL |
| pUC19 Control Plasmid, Linearized (Ampr, 40 ng/μL) | 5 μL |
| 500 bp Control Fragment (20 ng/μL) | 5 μL |
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