Aim: DNA molecular size markers or DNA ladders play a vital role in molecular biology laboratories where DNA electrophoresis experiments are usually conducted. This study aimed to produce a 100 bp DNA ladder at laboratory scale, which could be applied to determine the size of DNA fragments in molecular biology experiments. Methodology and results: In this study, 14 primers including 4 forwards and 10 reverses were designed based on the 16S rRNA gene sequence of Bacillus subtilis. These primers were able to amplify 10 DNA fragments with accurate sizes from 100 to 1000 bp. Furthermore, touchdown PCR was involved to maximize the specificity and yield of PCR products. Ten DNA fragments with the sizes including 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 bp were synthesized, and such bands were equivalent with commercial DNA ladders. Moreover, the quantity and quality of PCR products were measured using a nanodrop spectrophotometer. The optimal concentration ratios between such fragments (100- 1000 bp) were 800, 300, 150, 150, 500, 50, 50, 50, 50 and 50 (ng/μL), respectively. These ratios showed the clear and high resolution on 1.5% agarose gel. Conclusion, significance and impact of the study: The results indicated that 16S rRNA gene of B. subtilis was a potential material for DNA ladder preparation due to the multiple copies number of this gene. Furthermore, in combination with touchdown PCR, the nonspecific bands were reduced, and the products could be used directly without the need of purification step.