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From 2010.igem.org

Ligation

Materials:

- digested plasmid DNA or PCR product

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

- T4 ligase (Fermentas or BioLabs)

- H2O

- water bath at 16 °C

Protocol:

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