"

From 2013.igem.org

> iGEM Home

BioBrick Ligation

Supplies:

T4 DNA ligase

Ligase buffer

dH2O

Insert and vector

Steps:

1. Get ice.

2. Put the reagents on ice.

3. Calculate insert vector amount. Add the calculated amounts into eppendorf tube.

• Formula 1: insert mass(ng) = (1/6) x (insert length/vector length) x vector mass (ng)

• Formula 2: volume of insert(µL) = (total plasmid length/ insert length) x (insert mass/plasmid concentration)

4. Make a reaction mix with everything but the insert.

5. Aliquot the master mix into eppendorf tubes.

6. Add insert DNA.

7. Let incubate at room temperalerature for at least 2h or overnight.