How can I ensure transformation efficiency and overall cloning efficiency?
How can I ensure transformation efficiency and overall cloning efficiency?
In-Fusion Cloning is an all-in-one solution that maintains high transformation efficiency and also provides the highest possible level of cloning efficiency. While high transformation efficiency allows for a large number of transformed colonies, high cloning efficiency speaks to accuracy—ensuring that over 95% of transformants are correct, thus reducing the amount of time necessary to screen colonies.
-The primers must be of good quality to ensure the sequence of the homologous region is correct, allowing the cloning reaction to proceed efficiently and accurately.
-Clean PCR fragments are key for successful cloning. We recommend NucleoSpin Gel and PCR Clean?Up for your purification, which is included in some In-Fusion Snap Assembly bundles.
-For cloning efficiency, it is important that the PCR fragment be purified away from dNTPs and PCR primers after amplification.
-Use highly competent E. coli cells that have a transformation efficiency greater than 108 cfu/µg supercoiled DNA. Most homemade competent cells are not competent enough, especially if these cells are stored before use. We recommend Stellar Competent Cells, which are optimized for use with In-Fusion Cloning and are included in all bundles.