Notebook

Protocols

Ligation

After following our restriction digest protocol (which uses 250ng of DNA) you may follow these steps for ligation.
· Add 2µl of digested plasmid backbone (25 ng)
· Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 µl)
· Add equimolar amount of XbaI PstI digested fragment (< 3 µl)
· Add 1 µl T4 DNA ligase buffer. Note: Do not use quick ligase
· Add 0.5 ul T4 DNA ligase
· Add water to 10 ul
· Ligate 16C/30 min, heat kill 80C/20 min
· Transform with 1-2 ul of product
Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.