Cost-effective DNA sequencing and template preparation were evaluated for high-throughput screening of bacterial colonies. The rolling-circle amplification to generate template for DNA sequencing was carried out using 10-fold smaller amount of phi29 DNA polymerase and excluding two other enzymes (yeast pyrophosphatase and calf intestine alkaline phosphatase) used previously. Then, a 1/40 volume of the fluorescent terminator mix recommended by the manufacturer gave a usable sequencing result although a 1/26.7 volume of the mix was used to compare the protocol more accurately with manufacturer's protocol. (c) 2008, The Society for Biotechnology, Japan. All rights reserved.