The development of cost effective 100 base pair prototype DNA ladder using polymerase chain reaction

Background: In genomics, DNA scale is used as a standard unit for the measurement of unknown DNA fragments, plasmids, and PCR products during gel electrophoresis. The 100 base pair DNA ladder is essential and cost-effective in molecular biological research and is available commercially which is too expensive and not easily accessible to a common researcher for laboratory usage.Methods: The main purpose of this study was to report easily and practical method to prepare 100 base pair DNA ladder by simple PCR using pCAMBIA 1301 plasmid as a template which is an effective cost reduction strategy for laboratories. pCAMBIA 1301 was transformed into Escherichia coli (Top 10) bacteria by using heat shock method for high the yield of the plasmid. Bacteria containing our desire plasmid were cultured and plasmid was extracted from bacteria by using kit method. About 10 pairs of primers were designed from the backbone of the plasmid which amplifies 100 to 1000 base pair of PCR product with an interval of 100 base pair fragments. These fragments were optimized by using gradient thermo cycler and PCR products were purified using kit methods. For the stability of 100 base pair DNA ladder, it was placed in seven different buffers.Results: The outcome of this study shown that polymerase chain reaction was able to amplify 10 different types of DNA fragments which ranges from 100 to 1000 base pair with high qualification and size accuracy. PCR products were purified and sequenced. DNA ladder was pooled in seven different buffers and stored at -20°C. These buffers were used to optimize and evaluate the stability of the prototype DNA ladder.Conclusion: Our laboratory made 100base pair DNA ladder is very cost effective, it only cost 11 USD to prepare DNA ladder. This 100 base pair DNA ladder provides an independent quantitative unit that can be used with any biological application or technology, enabling genomes to be measured using a common metric.Keywords: 100 bp DNA ladder, pCAMBIA 1301 plasmid; PCR technique; Gel electrophoresis; Break Even Point Analysis