Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.
| Autor: | Yonemoto IT; J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA, 92137, USA., Weyman PD; J. Craig Venter Institute, Synthetic Biology and Bioenergy Group, 4120 Capricorn Lane, La Jolla, CA, 92137, USA. pweyman@jcvi.org. |
|---|---|
| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2017; Vol. 1498, pp. 359-366. |
| DOI: | 10.1007/978-1-4939-6472-7_24 |
| Abstrakt: | Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation. |
| Databáze: | MEDLINE |
| Externí odkaz: |
|