[Excerpt] Introduction: Rolling Circle Amplification (RCA) is an isothermal nucleic acid amplification technique that generates long single stranded DNA (ssDNA) molecules. RCA offers several advantages over other nucleic acid amplification techniques, such as Polymerase Chain Reaction (PCR), including thermal stability and high product yield from minimal DNA input (Demidov, 2002). In RCA, DNA polymerases bind to circular DNA templates, and continuously add deoxynucleotide triphosphates (dNTPs) to yield a long concatemeric strand of genomic material. Random hexamer primers can produce branched concatemers, whereas this study employed specific primers designed to bind a single location on the template. This approach yields a long, unbranched ssDNA strand. Puc19 was used in this study as the template plasmid, as it is well established as a model plasmid and is already circularized, negating the need for an additional circularization step. Once the unbranched single stranded concatemer is produced through RCA, another polymerase can be added to fill in the second strand, ultimately producing a long double stranded DNA (dsDNA) product. A large single stranded product was successfully obtained using the RCA protocol. This product was subsequently run with the New England BioLabs (NEB) Q5 Hot Start High-Fidelity PCR kit to create a double stranded product from the single stranded RCA product. After extension with Q5, the product displayed increased band intensity at the well and a higher measured concentration (Fig 4., Table 4). However, it remains unclear whether this product is double stranded, and additional analyses are required to confirm strand composition. This study aims to optimize the RCA reaction to yield a final end product of long concatemeric double stranded DNA that contains multiple copies of the template plasmid.