Generating single-stranded DNA circles with minimal resources

Single-stranded circular oligonucleotides are heavily utilized in rolling circle amplification and rolling circle transcription technologies. Although various reported methodologies are available to synthesize circular, single-stranded DNA (ssDNA), the unduly complicated protocols and the associated cost minimize the utility of these methodologies to a non-expert or a beginner in the field. Our protocol provides the simplest yet robust synthesis of circular ssDNA templates to be utilized in various applications, using minimal resources. • In this manuscript, we describe the most basic approach to synthesize circular ssDNA. • Our method utilizes the minimal resources, yet it is robust. • The utility of the methodology is very high for a non-expert or a beginner in the field.