The fact that your positive control worked rules out any issues with: thermocycler machine, PCR program settings, and any shared reagents like primers or polymerase.

That means the issue is unique to your DNA samples.

The first thing I would try is diluting your DNA samples. I know it sounds counter-intuitive, but dilution will help decrease any PCR inhibitors present in the DNA extraction (if you have any).

Try making 1:10, 1:100 dilutions of just one or two of your DNA samples & set up the PCR again. Make sure to include the positive control!

Good luck!

The reason for PCR failure is usually rapidly identified. Often problems can be explained by the fact that essential components like Mg2+ ions or even primers were of poor quality because the expiration date has passed or were unintentionally not added to the reaction mix.The PCR set-up is then started from scratch with new aliquots of components and in most cases the problem is solved. A well-accepted approach to reduce the risk of making an error in preparing the basic PCR mix is the utilization of a ready-to-use mastermix that contains all components except for the primers and the probe(s). These ready-to-use mixes have become popular since they contribute to a certain level of standardization and speed up and ease hands-on times.

Rohit Kumar , the success of the positive control shows that the failure is unique to the DNA samples and NOT due to an issue with any shared reagents (e.g. buffer) and NOT due to an issue with the machine.

Thanks for your replay

There are several reasons why PCR (polymerase chain reaction) may fail. some common causes are:

If you have good DNA quantity and quality and a positive control sample that worked, then the most likely reason for PCR failure in your experimental samples is related to issues with the primers or PCR conditions. Here are some possible explanations for your results:

To troubleshoot your PCR, you could try the following:

As it is recommended before by Burnette, I diluted the DNA to 1:10 to remove inhibitors, it work with one sample but not with the others (However they are tried before with the same marker and they work). What can I do? Do you think if I increase the annealing temperature I may get a PCR product? Any new advice? And what it is the original concentration of the DNA (before dilution) that it needs to be diluted to remove any inhibitors? There is a ratio between dilution percentage and the original concentration of the DNA?

as much i understand the PCR results depends on different things like

experience, positive/negative control, DNA amount, ramping rate of PCR, balancing of tube in PCR machine, annealing temp, primer concentration and no of cycles.

There are several reasons why a PCR reaction may fail, even when using good quality DNA and appropriate primers. Here are some possible reasons for your PCR failure:

To troubleshoot your PCR failure, you may want to try optimizing the reaction conditions or using a different polymerase enzyme. It may also be helpful to run a gel electrophoresis to confirm that there is no PCR product.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

Thanks for clarification. If DNeasy Plant Mini Kit is used for DNA extraction from seaweed samples, do you think that inhibitors (e.g. polyphenols, polysaccharides, and pigments) will be removed during the extraction? Or still we need to use a purification kit to remove those inhibitors?