Hi
Although the ladder is sharp but my pcr bands are too fuzzy and like smears. I try to set up one of my genes but it still stands for 3 bands, unfortunately. Tm is 65 but there are still nonspecific bands upper and lower of my specific band.
Could any one helps me with this?!
🙏 Thanks
Hi! It sounds like you're having trouble with your PCR results. 1. Sharp Ladder but Fuzzy Bands: The marker (ladder) used to compare your results looks clear and sharp, but your actual PCR results are blurry and smeared. 2. Multiple Bands: When you run the PCR for one of your genes, you're seeing three bands instead of one. This means there are extra, unwanted DNA fragments. 3. Non-specific Bands: Even though the melting temperature (Tm) for your PCR is set to 65°C, you're still getting bands that are not specific to your target DNA. These unwanted bands appear above and below the one you want. **Suggestions**: - 1. Optimize Annealing Temperature: Try adjusting the temperature to see if it reduces the non-specific bands. - 2. Improve Primer Design: Make sure your primers are specific to your target gene and don’t bind to other parts of the DNA. - 3. Check Reagent Quality: Ensure all your PCR reagents are fresh and properly prepared. - 4. Adjust Mg2+ Concentration: Sometimes changing the concentration of magnesium ions can help reduce non-specific binding. I hope this helps!
- Badges
- Science topic
- Similar topics
- Engineering
- Control Theory
- Control Systems
- Control Systems Engineering