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Background: Molecular DNA markers are one of the essential tools in molecular biology labs with varied applications. In the present study, we suggest an efficient and available strategy to produce molecular size marker in routine laboratories. Materials and Methods: To achieve the desired sizes of DNA fragments, we recruited PCR and bioinformatics techniques to synthesize 14 DNA fragments ranging from 100 to 3000 bp. Results: Holistic analysis of different parameters in primers design resulted in amplification of fragments in just one PCR program without any by-product and purification step. Our applied method enables researchers to modify amplified DNA fragments by wide range of chemical modifications toward varied applications. Conclusion: Method of home-made DNA ladder production by available ingredients and routine techniques reported in this study can be used in common laboratories for different applications.