Ligation
Procedure:
- Add 2μl digested plasmid backbone (25ng).
- Add equimolar digested fragment.
- Add 1μl T4 DNA ligase buffer.
- Add 0.5μl T4 DNA ligase.
- Add ddH2O to 10ul volume.
- Ligate at 16℃ for 30 minutes. Heat kill at 80℃ for 20 minutes.
- Transform 1-2μl of the product.