Cobia PCR of sex-specific markers

(c ephx1_1 ) and (c ephx1_2 )

20 μL reaction containing:

• 2.08 µL of 10X Taq Buffer

• 0.42 µl of dNTPs (10 µM)

• 0.67 μL of each primer (10 µM)

• 0.17 μL of Taq DNA polymerase (5 units/μL)

• 30 ng of extracted DNA

• made up to final volume with nuclease-free water

Thermal cycling:

• 3 mins at 94°C

• 30 cycles of 1 min at 94°C, 1 min at 60°C (c ephx1_1 ) and 61°C (c ephx1_2 ), and 1 min at 72°C

• 10 mins at 72°C

(c ephx1_1 )

20 μL reaction containing:

• 2.08 µL of 10X Taq Buffer

• 0.42 µl of dNTPs (10 µM)

• 0.67 μL of each primer (10 µM)

• 0.17 μL of Taq DNA polymerase (5 units/μL)

• 35 ng of extracted DNA

• made up to final volume with nuclease-free water

Thermal cycling:

• 3 mins at 94°C

• 30 cycles of 1 min at 94°C, 1 min at 66°C (c ephx1_1 ), and 1 min at 72°C

• 10 min at 72°C

(c ephx1_2 )

20 μL reaction containing:

• 2.08 µL of 10X Taq Buffer

• 0.42 µl of dNTPs (10 µM)

• 0.67 μL of each primer (10 µM)

• 0.17 μL of Taq DNA polymerase (5 units/μL)

• 20 ng of extracted DNA

• made up to final volume with nuclease-free water

Thermal cycling:

• 3 mins at 94°C

• 20 cycles of 1 min at 94°C, 1 min at 65°C (c ephx1_2 ), and 1 min at 72°C

• 10 mins at 72°C

20μL reaction containing:

• 2 µL of 10X Buffer

• 0.4 µL of dNTPs (10 µM)

• 0.8 µl of MgSO4

• 0.4 µL of each primer (10 µM)

• 0.08 µL of Taq DNA polymerase

(5 units/μL)

• 32 ng of extracted DNA

• made up to final volume with nuclease-free water

Thermal cycling:

• 2 mins at 94°C

• 30 cycles of 15 secs at 94°C, 30 secs at 62°C (c ephx1_1 ) and 62°C (c ephx1_2 ), and 1 min at 72°C

• 10 mins at 72°C

25μL reaction containing:

• 12.5 µL of Q5

• 1 µL of each primer (10 µM)

• 15 ng of extracted DNA

• made up to final volume with nuclease-free water

Thermal cycling:

• 30 secs at 98°C

• 30 cycles of 10 secs at 98°C, 30 secs at 61°C (c ephx1_1 and c ephx1_2 ), and 30 secs at 72°C

• 2 mins at 72°C