In this exercise, you will learn to perform a restriction cloning which will allow you to combine two elements from different plasmids together to form a new more desired product.
Learning Objectives
At the end of the activity, you will be able to:
Abilities
Utilize knowledge of restriction enzymes to determine which enzymes are best to use in a restriction cloning
Understand how to use ligase
Skills
Using Benchling's software to determine the best restriction sites for the cloning
Being able to preform a restriction cloning
Instructions
Now that we have amplified and purified samples of our mRFP and kan-backbone we can join them together through a ligation.
1. Go back to Benchling and look at the PCR product that was created and what are the best restriction enzymes to use if we want to create a sticky end ligation.
2. Digest your purified PCR samples with the correct enzymes using the following table as a guide
Solution | Amount | Notes |
DNA | ~500-1000ng | You want to have around 1000ng of DNA for very strong bands but sometimes your concentrations are too low so you can lower the quantity of DNA to ensure you have enough sample for all your lanes (you can get bands with even just 100ng of DNA) |
Enzyme | 1uL each | |
Buffer | 1.8uL | Use Benchling to determine what the best buffer for each enzyme is |
Distilled Water | Variable | Use Distilled water to bring total solution volume up to 20uL |
Total Volume | 20uL | ​ |
3. Place your digest in the 37 Incubator for 60-90minutes.
4. Once you have retrieved your digest samples from the incubator, prepare the following reaction mixture in a PCR tube
Solution | Amount | Notes |
Linear Vector DNA | 20-100ng | Larger Fragment that will makeup the backbone of the new plasmid |
Insert DNA | 1:1 to 5:1 molar ratio over vector | Smaller fragment that we will be adding to the backbone |
10x T4 DNA Ligase Buffer | 2uL | ​ |
T4 DNA Ligase | 5U | Look at the vial to determine how many U (units) per uL |
Water | Fill to 20uL | ​ |
Total Volume | 20uL | ​ |
3. Incubate for 10min at 22C (you can increase the time up to an hour to increase the efficiency)
4. You can now run your sample on a gel to see if it was successfully ligated. For this retrieve new PCR tubes and add 5uL of your ligation product to these new tubes. Add 1uL of loading dye to the lid of the tubes. Close the tubes and centrifuge to pull the dye through the solution. You can now add 6uL of your sample to your gel.
