To help life science research,
let more research results benefit mankind
To help life science research,
let more research results benefit mankind
Different from the traditional seamless cloning kit, the kit can easily achieve 2~6 fragments seamless cloning.
Highlights
1) Wide range: suitable for recombinant cloning of 1~5 fragments.
2) Quick: the reorganization can be completed within 5~60 minutes.
3) Simple: there is no need for fragment enzyme digestionit and not limited by the restriction of enzyme digestion sites;.
4) Seamless: no additional sequences are introduced.
Introduction
Uniclone One Step Seamless Cloning Kit is a simple, fast and efficient seamless cloning kit developed based on the principle of homologous recombination. It can recombine any DNA fragment containing the overlapping region of the end of the vector onto the linearized vector, which is not limited by the restriction of enzyme digestion sites, and has the low vector self ligation background. This method requires adding a 15-25 nt sequence at the end of the linearized vector to the 5’end of the forward / reverse PCR primer of the inserted fragment, so that the fragment and the vector can be recombined through the action of recombinant enzyme because of overlapping region.
Composition
Components | Amount(50 rxns) |
2×Uniclone Seamless Cloning Mix | 250 μL |
pUC19 Control Plasmid,Linearized(Ampr,40 ng/μl) * | 5 μL |
Control Fragment(500 bp,20 ng/μl)** | 5 μL |
* And ** respectively represent control linearized vector and control linearized fragment, these two components are connected with each other, which can eliminate the influencing factors of the experiment.
Related Products
Type | Composition | Cat. No. | Amount | Introduction |
Nucleic Acid Isolation | GE706 | 200 rxns | One box of dual-purpose, can effectively recover 80 bp ~10 kb double-stranded DNA fragments, can also directly recover PCR stock solution, gel without weighing, easy to operate, recovery rate of up to 85% | |
High-Fidelity PCR | SF212 | 5×1.125 mL | It is a 1.1 × ready-to-use mixture for efficient PCR amplification, which has high fidelity and extremely fast extension speed. | |
Plasmid DNA Extraction | PE707 | 200 rxns | Improved SDS alkali lysis method, high-purity plasmid can be extracted in 30 min |
Q1: No or Few Colonies Obtained from Transformation?
A1:
1) Low transformation efficiency. Bacteria were not competent. Check transformation efficiency by the control plasmid. You should obtain >1 x 108 cfu/μg; otherwise use fresh competent cells.
2) Transformed with too much/little reaction, see above protocol for details.
3) The PCR product was not purified or spent too much time under UV irradiation . It is recommended to be operated at 365 nm long wave UV.
4) Linearized vector and insert fragment are not pure and inhibit reaction. The experiment proves that EDTA and guanidine in products which were not fully purified can significantly reduce the recombinant efficiency. It should be purified again.
Q2: Large Numbers of Colonies Contained No Insert?
A2:
1) Incomplete linearization of your vector. It is important to remove any uncut vector prior to use in Sosoo reaction. If necessary, recut your vector and gel purify.
2) Contamination of Sosoo reaction by plasmid with same antibiotic resistance. If your insert was amplified from a plasmid, closed circular DNA may have carried through purification and contaminated the cloning reaction. Firstly, you must ensure the removal of any plasmid contamination, we recommend linearizing the template DNA before performing PCR. Secondly, If you spin-column purify your insert, treating the PCR product with DpnI before purification will help to remove contaminating template DNA.
Q3: Clones Contained Incorrect Insert?
A3:
1) Your PCR product contained non-specific sequences. If your PCR product is not a single distinct band, then it may be necessary to optimize your PCR protocol or gel purify the PCR product to ensure cloning of the correct insert.
2) Polluted by plasmid with same antibiotic resistance. If your linearized vector is not obtained by empty vector but by the vector that had been inserted into the other fragments , it may result in the some background and make some clone contains incorrect insertion fragment. Improve the efficiency of enzyme, treating the PCR product with DpnI before purification, using adbanced linearized vector as PCR template all can effectively avoid such situation happen.
Manual: Click to download
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