You have a lot of primer dimer so your primers are annealing together and not amplifying your dna. It is possible that your dna either has a polymorphism at the 3'end of one primer which is stopping amplification and allowing the primer dimer to form so I would try

1 Use a new DNA that has amplified before with other primers and repeat the gradient....your dna may be too concentrated or contain pcr inhibitors....how much dna are you amplifying?

2 use half as much primer as too much primer can help P-D formation. 3 Use a hot start Taq which will stop P-D formation.

4 Using another working set of primers amplify a DNA that works with the other primer set and add your DNA from this picture to the reaction mix. If doing this makes the reaction fail then you may have pcr inhibitors in your dna sample.

5 drop your pcr volume to 25ul. A smaller volume reaches temperatures quicker and may work better

First, the bottom bands would be primer dimer, sometimes the denaturing step is not enough so you can heat the work primer stock at 95 C for 3 min before mixing them (heat them and cool down on your bench before adding to your mix ).

2. You would try a small volume reaction of 15 ul for each annealing temperature.

3. I can see a high molecular weight band (at the right part), you would reduce the DNA concentration (1-10 ng total should be ok ).

Good luck

Noe

First, the bottom bands would be primer dimer, sometimes the denaturing step is not enough so you can heat the work primer stock at 95 C for 3 min before mixing them (heat them and cool down on your bench before adding to your mix ).

2. You would try a small volume reaction of 15 ul for each annealing temperature.

3. I can see a high molecular weight band (at the right part), you would reduce the DNA concentration (1-10 ng total should be ok ).

Good luck

Noe

I would start with extending your initial 95C heating step to 2-3 minutes, rather than one. You could also try extending the annealing step just a little, to 40 seconds, and keep exploring the best temp for annealing (our lab uses anything from 50-66 C).

Other things to check would be the molarity of your primers (10 uM is typical for PCR), and the purity of your starting DNA.

It's always possible that you have a primer that doesn't work with your sample, for whatever reason. If you have another set of primers compatible with your sample, you can try them out to see if you can get a product without changing anything else--then you will know it's a primer issue!