Gibson Assembly® is a standard technique in synthetic biology used to assemble multiple DNA fragments into a linearised plasmid vector.
In order to successfully obtain an assembled plasmid, the amounts of DNA fragments and the plasmid used must have precise ratios.
Calculating the exact volumes of resuspended DNA fragments and plasmid needed to achieve this ratio can be quite time-consuming, especially if many assemblies need to be performed.
Therefore, we have developed a web application that allows scientists to quickly obtain the volumes for the assembly of up to 6 fragments.
HOW TO USE:
1) Select the number of DNA fragments to assemble together (the plasmid is regarded as a fragment and should be included in the count).
2) Choose which variable to control between:
- Final reaction volume
- Final amounts of DNA in the reaction
New England Biolabs protocol provides suggested total DNA amounts depending on the number of assembled fragments. However, in order to obtain the desired amounts, many times reaction volumes become extremely large. This is quite a drawback since it wastes a lot of reagents.
By tweaking these two parameters you can optimize your reaction conditions to either obtain a set final DNA amount or a set final reaction volume.
3) Select the desired value for the controlled variable
4) Choose the molar ratio between DNA fragments and plasmid
Sometimes an excess of fragments is needed in your reaction mixture for the assembly to work.
With this option the ratio of fragments to plasmid can be set without modifying the other parameters such as reaction volume or DNA amount
4) Enter the concentrations (ng/uL) of the DNA fragments
5) Enter the lengths (bp) of the DNA fragments
6) Calculate!
Number of fragments
[including linearized vector]:
Fragments:Plasmid Ratio:
