Sequencing reaction with Big Dye Terminator v 3.1 (ABI)

After treatment with Exonuclease I and Shrimp Alkaline Phosphatase add 8 ul of deionized water to reaction, and mix by pipetting up and down. Transfer 3 ul aliquot of diluted reaction mix into a semi-skirted 96-well PCR plate.

1x sequencing reaction mix

3 ul purified and diluted PCR product

2 ul sequencing primer (1.6 pmole/ul)

1.5 ul 5x sequencing buffer

1 ul 50% DMSO

1 ul Big Dye v 3.1

1.5 ul deionized water

Cycling conditions for sequencing reaction:

98 °C – 5 min

40 cycles of

96 °C – 10 sec

50 °C – 5 sec

60 °C – 4 min