Gibson assembly preparation

For the Gibson assembly you need DNA fragments with overlapping ends. These fragments are often obtaind by a restriction digest with the matching restriction enzymes.

To obtain high yeilds of the Gibson product the same number of each DNA fragment should be added to the reaction mix. In most cases the fragemnts have different lenghts, therefore you need to calculate the equimolar concentration with the following formula:

The concentration of the different DNA fragments can be measured using Nanodrop, which is an instrument that allows measuring nucleic acid concentrations in a minimal amount of liquid. The length of the fragemnts can be deterimed from the plasmid maps. Once you calculated the amount needed from each fragment you have to add water up to to the predefined reaction volume. To start the reaction you only need to add the same volume of 2X Gibson reaction mix and incubate the sample at 50 degrees Celsius for one hour.