I am performing PCR on environmental DNA samples collected from water to detect mussels. While our positive controls (tissue and swab samples) consistently yield strong bands, none of our eDNA water samples show amplification, even though we know that they contain mussel DNA.
We have tried several approaches to resolve the issue, including:
- Varying the number of PCR cycles
- Using different commercial kits for inhibitor removal
- Testing different input volumes of DNA
Despite these adjustments, we still only see amplification in the positive controls.
We are using Phusion Plus Master Mix and the following PCR cycling protocol: initial denaturation at 98 °C for 30 seconds; 40 cycles of 98 °C for 10 seconds, 60 °C for 10 seconds, and 72 °C for 15 seconds; followed by a final elongation at 72 °C for 5 minutes and a hold at 4 °C.
This protocol consistently produces clear bands for the positive controls.
Has anyone experienced similar problems or have suggestions on what could be the cause?
Hi Victor,
for me it sounds that you have either low template concentrations or compromized template (I don't now how you check this) because you rpositive controls seem clearly to work. If you don't get a PCR product due to low template concentration there are a few thinks you can try:
1. Increase cycle number
2. Optimize Annealing temperature (do a gradient PCR if possible) - it might be that your annealing is sufficient when you have relative high template concentration but not suffiecient enough for low template concentration
3. Increase primer concentration
4. Try additives like DMSO or BSA - they can help to improve specifity and yield
5. Optimize magnesium chloride concentration
6. if none of the above works I would try nested PCR - this often helps when you have difficulties due to low template concentrations.
Good luck
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