May 20, 2024
- 1James Cook University
- GigaScience Press
External link: https://doi.org/10.1093/gigascience/giae034
Protocol Citation: Zhi Weng Josiah Poon 2024. Cobia PCR of sex-specific markers. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q7r3l1y/v1
Manuscript citation:
Shen X, Hu J, Yáñez JM, Bastos Gomes G, Poon ZWJ, Foster D, Alarcon JF, Shao L, Guo X, Shao Y, Huerlimann R, Li C, Goulden E, Anderson K, Fan G, Domingos JA. Exploring the cobia (Rachycentron canadum) genome: unveiling putative male heterogametic regions and identification of sex-specific markers. Gigascience. 2024 Jan 2;13:giae034. doi: 10.1093/gigascience/giae034.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 19, 2024
Last Modified: May 20, 2024
Protocol Integer ID: 100091
Abstract
PCR for sex-specific markers in Cobia (Rachycentron canadum)
Panama population (Taq PCR Core Kit (QIAGEN))
Panama population (Taq PCR Core Kit (QIAGEN))
(cephx1_1) and (cephx1_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 (cephx1_1) and 61°C (cephx1_2), and 1 min at 72°C
- 10 mins at 72°C
Brazil population (Taq PCR Core Kit (QIAGEN))
Brazil population (Taq PCR Core Kit (QIAGEN))
(cephx1_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 (cephx1_1), and 1 min at 72°C
- 10 min at 72°C
(cephx1_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 (cephx1_2), and 1 min at 72°C
- 10 mins at 72°C
Australia population (PlatinumTM Taq DNA Polymerase High Fidelity (Invitrogen))
Australia population (PlatinumTM Taq DNA Polymerase High Fidelity (Invitrogen))
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 (cephx1_1) and 62°C (cephx1_2), and 1 min at 72°C
- 10 mins at 72°C
Japan population (Q5 High-Fidelity 2X master Mix)
Japan population (Q5 High-Fidelity 2X master Mix)
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 (cephx1_1 and cephx1_2), and 30 secs at 72°C
- 2 mins at 72°C