Cost effective DNA sequencing and template preparation from bacterial colonies and plasmids
2009
Maruyama, Fumito | Nozawa, Takashi | Aikawa, Chihiro | Sakurai, Atsuo | Nakagawa, Ichiro
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.
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