Team:TU Delft/protocols/ligation




- digested plasmid DNA or PCR product

- T4 ligation buffer (10x) (Fermentas or BioLabs)

- T4 ligase (Fermentas or BioLabs)

- H2O

- water bath at 16 °C


Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier.

Reaction for one sample:

DNA insert x μL
DNA vector x μL
T4 Ligation buffer (10×) x μL (for 1×)
T4 Ligase 1.0 μL
H2O x μL
10-15 μL

The final concentration is preferably ~100 ng/μL. Incubate at 16 °C for at least 3 hours. For transformation use circa half of the ligation mix.

Transform circa half of the ligation mix. Incubate at 16 °C o/n