IGEM:British Columbia/Protocols/Biobrick Ligations

Biobrick Ligations

Materials

• T4 DNA Ligase
• Ligase buffer (1uL aliquots)
• sdH2O
• Insert and vector DNA (of approximately known concentration)

Equipment

• Vortex
• 16°C water bath

Method

• Calculate insert/vector amounts. Various ratios have been recommended, most commonly 3:1 or 6:1. Formula: insert mass (ng) = ratio x (insert length/vector length) x vector mass (ng)

Example: For a 150bp insert and a 2200bp vector, at a ratio of 6:1: insert mass = 9/22 * vector mass. If you have a measurement of 30ng/uL for the vector and 5ng/uL for the insert, you need 2.5uL of insert and 1uL of vector.

• Add 1uL ligase buffer (vortex, and make sure it still smells like wet dog).
• Add sdH2O to 9.5uL and vortex.
• Add 0.5uL T4 ligase.
• Incubate at 16°C for at least 1 hour (leave overnight after transforming in case it needs to go for a bit longer).