I have conducted a sexing PCR of several samples of avian tissue.The quality of the extracted DNA was good for all samples and no contamination was detected in the control sample.
For the PCR I used published primers and a tested PCR cycle. After gel electrophoresis, all of the samples produced bands at the expected sizes. However, in one of the samples, the heavier band (650 bp) is very very faint, while the lighter band (500 bp) is clearly visible.
All information I could find on faint bands in PCR was about optimizing the PCR itself, but since all other samples (that were amplified the same way at the same time) and even the lighter band of the same sample are clearly visible, I don't see how the PCR could have been the issue.
It is also unlikely that a bit of the other samples got into the wrong well, because the band that is faint should be visible in all samples.
What are other possibilities for this error?
Best regards,
Anne
Hey there,
other problems I can image could include:
- less input material
- a possible SNP making the primer binding less efficient
- less input primers
Hope this helps.
Regards,
André
Hello André,
Thank you so much for your answer! I will look into your suggestions.
Best regards,
Anne
Sometimes if bands of higher length are faint that is an indicator of secondary structures inhibiting full synthesis. Sometimes adding denaturing such as formamide to the PCR will help. There are many other denaturing agents and enzyme stabilizers that can help. Read the article "Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and
Optimization Strategies" for more details.
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