Hi,

I could see some primer dimer bands at the bottom of your gel.

Could you please share some additional details:

1. Have you included Positive control in your PCR reaction?

2. Have you checked primer concentration after re-suspension of your primers?

3 What is the expected amplicon size?

4. Have you tried primer blasting your designed primers?

5. What was the concentration of DNA / RNA used for the PCR?

Regards,

Ajith

Ajith Kumar is correct much more detail is needed as there are many reasons the pcr may fail. DNA quality is important.How much dna ,from what sourse and what od260/280 are you using. The annealing temp of the primers and the cycle conditions would help. Can you borrow a good dna sample from another researcher that has your gene in it and has amplified with other primers before so that uou can use it as a positive control to show that the pcr is working?. Good gel and size standard separation though while you are setting the pcr up run the samples less far so that any weak bands that you are getting become more visible just until things are working well. I do agree with @ajith that the primer dimer is much too strong. There is a lot of fluorescence for such a shoth molecule so try using much less 1/4 to start with primer but ideally if your lab has some use a hot start taq polymerase . The primer dimer is using up reagents so the correct pcr is working badly

Saied Mehdi Miri...quite many factors could be responsible for this, I suggest you avoid contamination in the gel electrophoresis prep and ensure the PCR conditions are monitored using a RT PCR method and appropriate controls that covers a wider amplification of the target DNA..e.g a multiplex if applicable to what you are trying to achieve..