Cycle sequencing

STeP method (Platt et al. 2007)

I recommend this method too fast (cycle sequencing finished only 55 min!).

Big Dye Terminator v 3.1 condition (Total 10 μL volumes)

BigDye Terminator v3.1 ... 0.5 μL

5× Sequencing Buffer ... 2.0 μL

Primer ... 2.0 μL (20 ng)

DNA ... 2.0 μL (200 ng)

Deionized Water ... 3.5 μL

<STeP cycle>

96 °C 01:00 s

96 °C 00:10 s |

50 °C 00:05 s | 15 cycle

60 °C 01:15 s |

96 °C 00:10 s |

50 °C 00:05 s | 5 cycle

60 °C 01:30 s | Δ15 s/cycle

96 °C 00:10 s |

50 °C 00:05 s | 5 cycle

60 °C 02:00 s |Δ45 s/cycle

finished

Platt, A. R., et al. (2007). Improved DNA sequencing quality and efficiency using an optimized fast cycle sequencing protocol. Biotechniques 43(1): 58-62.

Purified DNA and HiDi reaction

After finished cycle sequencing, add 100 % EtOH (100 μL), and CH3COONa (4 μL) each samples

centrifuge 130x100 rpm, 15 min

discard EtOH

add 70 % EtOH (200 μL) each samples

centrifuge 130x100 rpm, 15 min

discard EtOH

air dry

add 15 μL of HiDi and move to thermal cycler

98 °C 01:00 s

move to ice

finished