Might be possible there is some issue with your primers' specificity. consider checking your primers' efficacy in UCSC browser's In silico PCR. Moreover, check for the quality of your DNA, might be possible there are some contaminants challenging PCR amplification. also you need to optimize your primers manually through gradient PCR. It is always a good way to be sure of primers' annealing temperature.

1000 bp isn't very large. Did you try a gradient of annealing temps? Have you been able to amplify other regions from this DNA to make sure the template isn't degraded?

Make sure you're interpreting the Tm calculator correctly. You want your annealing temp to be about 5*C lower than the Tm.

Good luck!

Thank you both a lot for thinking along!

I already checked the primers in UCSC browser's In silico PCR and it shows the correct fragment, so that should not be the problem. I also tried an annealing temperature gradient, which did not show a band. Moreover, I also have amplified other regions of this DNA without a problem.

Does anyone else have another idea that might help?

try amplifying in final concentration of betaine 1molar and 5%dmso ( up to 8% is ok with pcr) using primers far apart then if this looks poorly amplified dilute the amplified product 1:100 and re amplify with any primer pair inside the first pair of primers also using betaine and dmso. Check the quality and quantity of your original dna sample in case it contains pcr inhibitors and try amplifying double dilutions ( 1/2, 1/4, 1/8 etc) as sometimes diluting a pcr inhibitor means better amplification even though there is less dna