|After growing up a part from a distribution or request, you'll want to miniprep it. At iGEM HQ, we use the following protocol.|
After following our restriction digest protocol (which uses 250ng of DNA) you may follow these steps for ligation.
- Add 2ul of digested plasmid backbone (25 ng)
- Add equimolar amount of EcoRI-HF SpeI digested fragment (< 3 ul)
- Add equimolar amount of XbaI PstI digested fragment (< 3 ul)
- Add 1 ul 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.