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.