You can determine the success of your restriction digestion by analyzing the resulting DNA fragments using various methods.
1. Gel Electrophoresis
- Visual Inspection: Running the digested DNA on an agarose gel allows you to visualize the fragments.
- If the restriction enzyme cut the DNA at the expected sites, you'll see distinct bands corresponding to the predicted fragment sizes.
- If the digestion was unsuccessful, you'll likely see a single band representing the uncut DNA or multiple bands that don't match the expected sizes.
- Size Comparison: Comparing the band pattern to a DNA ladder with known fragment sizes helps confirm the success of the digestion.
2. DNA Sequencing
- Verification of Cut Sites: Sequencing the digested DNA allows you to confirm the presence or absence of the expected restriction enzyme cut sites.
- This method provides definitive evidence of successful digestion by revealing the exact sequence of the DNA fragments.
3. Ligations and Cloning
- Successful Ligation: If the restriction digestion was successful, the digested DNA fragments will be able to ligate into a vector, resulting in the formation of recombinant DNA molecules.
- Transformation and Selection: These recombinant molecules can be transformed into competent cells, and the successful transformants can be selected and analyzed.
4. PCR Analysis
- Confirmation of Fragment Presence: PCR using primers flanking the restriction enzyme cut sites can be used to confirm the presence of the expected fragments.
- The absence of a PCR product indicates unsuccessful digestion.
5. Other Techniques
- Southern Blotting: This technique can be used to detect specific DNA sequences in the digested DNA sample, providing further evidence of successful digestion.
By employing these methods, you can confidently assess the success of your restriction digestion and proceed with subsequent experiments.
