Construction of Recombinant DNA Plasmid Using pUC18 for Production of DNA Ladder

Tin, Kok Chon (2023) Construction of Recombinant DNA Plasmid Using pUC18 for Production of DNA Ladder. Final Year Project (Bachelor), Tunku Abdul Rahman University of Management and Technology.

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Abstract

The DNA ladder is vital for molecular work as it allows the determination of size of a DNA fragment by acting as a size reference during gel electrophoresis. In this project, we planned to construct a DNA ladder plasmid since it allows for the rapid production of a significant amount of DNA ladder. The use of this construct enables generation of a standardized DNA ladder of specific fragment sizes for reproducible results and leads to long-term cost reduction. Here a strategy was devised to produce a 2000 bp DNA ladder by cloning multiple 250bp fragments into pUC18. To do that, a 250 bp amplicon was amplified from pUC18 and cloned into pUC18 to produce an intermediate clone, Intermediate I. However, neither cloning a second 250 bp fragment into partially digested Intermediate I nor cloning ligated multiple 250 bp fragments into pUC18 yielded viable transformants. We further repeated the above experiments in pBR322 instead of pUC18 and the same results were obtained. Since pUC18 is a synthetic plasmid derived from pBR322, we speculate that the fragment being cloned may result in plasmid loss from the host cell, thereby preventing the growth of the transformant on the selection medium. Keywords: DNA Ladder, pUC18, pBR322, EcoRI, PCR, Cloning