Produkty
Producenci
NZYEasy Cloning & Expression kit XVII
nr kat.:
MB33001
Opakowanie:
op.
Producent:
nr kat.:
MB33001
Opis
NZYEasy Cloning & Expression kit XVII was designed to allow directional cloning of any PCR-generated fragment or synthetic gene into the linearized pHTP17 Escherichia coli expression vector (contains a Ruminococcus flavefaciens cellulosomal protein, CpB, as fusion tag to promote solubility and folding). Cloning proceeds in a single ligase-independent reaction mediated by the NZYEasy enzyme mix.
Vector-complementary overhangs containing a specific sequence recognized by the NZYEasy enzyme are incorporated in the PCR product by using primers with appropriate 5’ extensions. When you combine the insert thus generated with the linearized pHTP17 vector, also containing complementary overhangs, in the presence of NZYEasy enzyme mix, the two DNA molecules will anneal through base-pair complementation of the single-strand regions.
The reaction occurs in a single-tube along three temperature-dependent steps. Circular recombinant vector containing the fragment of interest is obtained by transforming the annealed plasmid DNA into competent E. coli cells. The system allows achieving high cloning efficiency (80-100%) and does not require the use of DNA ligases. In addition, no further treatment (e.g. restriction digestion, phosphorylation, or blunt-end polishing) of the inserts is required.
Cloning is performed using conventional E. coli strains. Once pHTP17 recombinant plasmid has been constructed and its sequence confirmed it should be used to transform λDE3 E. coli lysogens, such as BL21(DE3), for high levels of protein expression.
This kit has been successfully used in high-throughput (HTP) platforms for the efficient cloning and expression of a large number of genes at a scale compatible with the functional screen of hundreds to thousands of genes/proteins.
Features:
- Ligase-independent cloning
- Time-saving and cost-effective method
- No additional treatment of the PCR fragments required (e.g. restriction digestion, phosphorylation or blunt-end polishing)
- High cloning efficiencies
- Directional cloning
- Possibility to clone multiple inserts simultaneously into one vector
- Compatibility with automation and high-throughput methods
- Suitable for both cloning or bacterial protein expression
- Recombinant proteins are expressed in fusion with NusA that promotes solubility and folding
Applications:
- Cloning of blunt-end or A-overhangs PCR products
- Bacterial protein expression (Escherichia coli)
Components:
- 10x Reaction Buffer
- NZYEasy enzyme mix
- Linearized ector
- Positive control
Manual for Multiple Fragment Cloning using pHTP vectors
NZYEasy Cloning Expression kits Product Brochure
FAQ NZYEasy clonning and Expression
Dane techniczne
| Opakowanie | op. |