I agree with Yuan-Yeu Yau

So, according to your explanation, this will come down the DNA sample you use. Probably different amount of sample was loaded into the PCR tubes (uneven pippetting results; DNA sample stuck to PCR tube wall by accident)? Probably the stock DNA sample is not homogeneous? For example, DNA pellet was not re-suspended to homogeneous?

I agree with Yuan-Yeu Yau

I'm following

i guess the DNA samples have different concentration or purity

have you tapped the DNA template before making the final reaction? to overcome with this problem always gentle tapping (2-3 times) before making the final reaction and pipetting error is also one reason because the template volume is a very small in amount so when you take each constitute, cross-check with manually for every reaction.

I hope this will help.

You can get this result if one primer of the poorly amplified product sits over a polymorphism so primer binding is reduced. Alternatively if one amplimer is GC rich and easily forms a stable conformation hiding the primer binding site or even just re anneals quickly with itself then pcr efficeincy is reduced. Possible also is primer degradation if the poor primers were old or have been stored in water not TE .

I think this is with pipetting error while adding templet.

OR

Degradation of your primer and templet.

DNA temples from which sample? What was the DNA lysate method you used?

Did you mixed equal loading dye in well of the gel?

Can you share your DNA quantification result? Did you dilute your sample?

Hi,

Please repeat the experiment with same input volume of DNA.

1.Most likely pipetting error

2. Both PCR used same primers or amplifying different genes?

Hope this helps.