We use Gibson assembly to assemble DNA fragments into plasmids. It is a multistep process, requiring first a PCR to create each fragment by copying them out of template plasmids, then a single isothermal assembly to stick the fragments together, then transformation into competent cells and culture for amplification.
Protocol adapted from Nikolaus Obholzer (https://wiki.med.harvard.edu/SysBio/Megason/IsothermalAssembly)
- Take the different DNA parts you want to assemble and measure their concentration with Nanodrop
- We need equal molecuar ratios! Divide the concentration measured above [ng/ul] by the length of each part to have equivalent numbers
- Based on these numbers, calculate the ul you need of each to have the same amount of molecules
- Take 5 ul of the mixed solution and add 15 ul of Gibson mastermix
- Heat at 50°C for 45-60 minutes (there is a programm written in the iGEM folder)
Now the samples are ready to be transformed.
PS: if someone has a less complicated method, please change the protocol :)