Have you checked the gDNA template sample on agarose gel to assess its integrity?

How long is your amplimer and how much dna are you using and in what volume pcr?

Paul Rutland my amplimer length is 1000bp , I'm using 200ng gdna as template for 10ul reaction.

try running 3 pcr reactions of 20ul volume and amplify 20, 10 and 5ng of dna. 20ul is more forgiving of small changes in conditions and too much dna can contain pcr inhibitors thar remove Mg ions and the reaction fails

Ahmad Hafiz Murtadha I did ! And I got a single crisp band closer to the well.

Paul Rutland I'm using cloning primers. Should I use primers without restriction flanks instead ?!

Geetha V C you need the cloning primers to work so I would use these primers. The thing that you have to remember is that for the first 2 cycles (and then the rest of the cycles) you mst calculate the Ta of the primers only for the length of the primer that anneals to the dna template not the full length of the primer

@Paul Rutland still no band 🥲

Are you getting a small ( about 100bp blurred band) indicating a primer dimer problem.? If you already have the non cut site primers you could try those in case the cut site primers are degraded...are they fresh and dissolved in TE not water? It may be a secondary structure problem with the template dna in which case trying amplifications with 1M final concentration of betaine and also try 2 pcrs with 5% and 8% dmso and also try an amplification with any other primer set as a test of the dna integrity. If all of these fail then trying a different polymerase can sometimes help Geetha V C

Is your initial denaturation at 95 °C for 5 minutes? Maybe using 5 to 10 minutes will help separate the gDNA better.