You could also use the Gibson Assembly Cloning Kit (NEB #E5510) to generate your construct. The advantage of Gibson Assembly is that it does not depend on restriction enzyme sites and so it can be used in all cases, regardless of the presence or absence of restriction sites in your target gene or vector. Please refer to https://www.neb.com/e5510 for and the IMPACT Frequently Asked Questions (FAQs) for further details.
The outline of the Gibson Assembly Cloning is given below:
- Design primers to amplify target gene (and/or vector) with appropriate overlaps. Try our primer design tool, NEBuilder™.
- PCR amplify target gene using a high-fidelity DNA polymerase. Prepare linearized vector by restriction digestion or PCR.
- Confirm and determine concentration of fragments and linearized vector using agarose gel electrophoresis, a NanoDrop™ instrument or other method.
- Add PCR fragments and linearized vector to Gibson Assembly Master Mix and incubate at 50°C for 15 minutes.
- Transform into NEB 10-beta Competent E. coli.
For more information on transformation and competent cell selection please refer to our website: http://www.neb.com/nebecomm/tech_reference/competent/default.asp