Cloning Insert into Backbone
Cloning
1. First digest the plasmid containing the insert as well as the backbone with the correct restriction enzymes that will place the insert in the correct orientation and location in the backbone. Recommended to do this step for longer than an hour.
2. Use gel electrophoresis to separate the insert and backbone from the rest of their respected plasmids.
3. Using QIAEX 2 Agarose Gel Extraction Kit (see to the right), extract the insert and backbone from the agarose gel.
4. Create Ligation Mixture as follows:
- 1 μL - T4 DNA Ligase Buffer
- 0.5 μL - T4 DNA Ligase
- 1 μL - Backbone DNA
- Insert added so that the insert and backbone have a 3:1 molar ratio
- ddH2O added to create a 10 μL Ligation reaction
5. Create a Ligation (-) control mixture:
- 1 μL - T4 DNA Ligase Buffer
- 0.5 μL - T4 DNA Ligase
- 1 μL - Backbone DNA
- 7.5 μL - ddH2O
6. Transform the entire ligation reaction into bacteria following the bacteria transformation protocol ensuring to complete the digestion step with the same enzymes used to clone the insert into the backbone to check if the cloning process was successful.
