In this exercise, you will learn to perform an in lab digest in order to learn how combining restriction endonuclease digestion is used to confirm that a plasmid DNA contains the expected endonuclease recognition sequences at the expected locations.
Learning Objectives
At the end of the activity, you will be able to:
Abilities
Set up multiple restriction endonuclease digest reactions efficiently.
Skills
Using Benchling's software to run a virtual digest of plasmids
Being able to perform an in lab digest
Instructions
1. Use Benchling to predict the fragments generated following digestion with EcoRI alone, with PstI alone, with EcoRI and PstI together, with EcoRI and XbalI together and with NotI alone.
2. Label four 0.2 ml PCR tubes 1, 2, 3,and 4.
3. Calculate the volumes of each solution you will be adding to your reactions using the following table as a guide. To calculate the volume of DNA you will be adding use your concentrations from mini-prep to guide you.
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 | ​ |
4. Retrieve the required enzymes and buffers from the -20 freezer and place them in an ice bucket with ice.
5. Set up the restriction digest reaction and place them in a 37 incubator for 60-90 min. Be extremely careful not to cross-contaminate the enzyme and buffer solutions. No double-dipping!
In this exercise, 15.2 uL of sample DNA was used, therefore the single digests had a total volume of 17.5uL and the double digests had a total volume of 18uL. 4uL of the DNA ladder was used in each of the corresponding gel wells (A and G). Note that you don't need to incubate the later nor add any dye --- pipette it straight from the stock solution to the gel wells.
Virtual digest of some of the enzymes found in the previous table.
Example 1. Results from a gel done in lab based on the previous virtual digest. The DnpI digest was complete, but the intensity was low because the DNA is spread over many bands. The middle lane shows an incomplete digest, and the third lane was deformed mechanically (i.e.: there is some bleeding of the adjacent DNA ladder).
Example 2. Another digest-- this time with EcoRI and PstI. In this case, both digests were complete and match the virtual digest done beforehand on Benchling. The intensity is high due to the high initial concentration of the plasmid sample used.
