Division 7 Sequencing of PCR products

DNA shall be sequenced using the Big Dye Terminator Kit v3,1 (Applied Biosystems). The total volume in each reaction is 20 μl, the final concentrations being 1 × Big Dye terminator, 1 × sequencing buffer, 10 pmol/μl forward or reverse primer and 10 μl of purified DNA (diluted to approximately 10 ng/μl) run using the following cycling conditions on a DNA Engine Tetrad 2 Peltier Thermal Cycler (or equivalent): 94 °C for 30 seconds followed by 96 °C for 10 seconds, 50 °C for 10 seconds and 60 °C for four minutes, cycling the last three steps 30 times.

The PCR products shall be precipitated using a sodium acetate method where 20 μl DNA is added to 10 μl NaAc, 70 μl H₂O and 250 μl ethanol, vortexed and centrifuged at 13 000 rpm for 20 minutes, the supernatant shall be removed and the pellet shall be washed with 200 μl absolute ethanol, centrifuging at 13 000 rpm for five minutes. The pellet shall be dried for five minutes at 37 °C. 25 μl of Hi-Di formamide shall be added to the pellets, heated to 95 °C for two minutes and vortexed thoroughly. Samples shall be sequenced using the ABI3130xl Avant Genetic analyser according to manufacturers' instructions. Sequencing results shall be analysed using Sequencher software and sequences matched to sequences on the NCBI database using the BLAST function.