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.