Seamless DNA Assembly Plus Kit
Based on 4 publication(s) in Google Scholar
MCE Seamless DNA Assembly Plus Kit contains an optimized mix of recombinase, reaction buffer, and additional cofactors that significantly improve the cloning efficiency and tolerance to impurities. This product can complete multiple DNA fragments recombination and takes only 5 minutes for single fragment, and the positive rate is more than 95%.
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Storage :
Store at -20°C for 2 years. Avoid repeated freeze-thaw cycles.
To place orders, for customer services and technical support, please contact: MedChemExpress USA
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Agarose With TAE Powder (1%)
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 Plus Kit contains an optimized mix of recombinase, reaction buffer, and additional cofactors that significantly improve the cloning efficiency and tolerance to impurities. This product can complete multiple DNA fragments recombination and takes only 5 minutes for single fragment, and the positive rate is more than 95%.
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 Assembly Mix Plus | 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 |
|---|---|---|
| Single insert fragment | 50°C | 5 min |
| 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
Store at -20°C for 2 years. Avoid repeated freeze-thaw cycles.
Components
| Components | HY-K1041-50 rxns |
|---|---|
| Seamless Assembly Mix Plus | 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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