BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and ABI PRISM 310 automated sequencer (Applied Biosystems, USA) used to detect the species specific sequencing regions of PCR products according to the manufacturers’ instructions. In brief, a master-mix was prepared in a tube and mixed by a vortex machine. A volume of 2.0 μL of 5X sequencing buffer and 1.0 μL of BigDye ready reaction mixture and 0.32 μL of individual primer (forward or reverse), 3.5 μL of PCR products and 13.18 μL of nuclease-free water was added into the volume and make 20 μL in total. Samples were vortexed and centrifuged and then PCR strip was placed in a cycle sequencing machine (Mastercycler® gradient, USA Scientific) and the PCR cycling profile were pre-denaturation at 94 °C for 1 min; 25 cycles of denaturation at 94 °C for 10 s, annealing at 58 °C for 5 s and extension at 60 °C for 4 min; and a final extension at 60 °C for 10 min. The tubes were subjected to capillary electrophoresis through (denaturing) POP-6TM polymer sequencing machine (ABI PRISM® 310 Genetic Analyzer).
Bigdye chain terminator version 3.1 cycle sequencing kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BigDye Chain Terminator version 3.1 Cycle Sequencing Kit is a DNA sequencing reagent kit developed by Thermo Fisher Scientific. The kit contains the necessary components for performing dye-terminator cycle sequencing, a method used to determine the nucleotide sequence of DNA samples.
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3 protocols using bigdye chain terminator version 3.1 cycle sequencing kit
1
DNA Sequencing Using BigDye Terminator
2
PCR Product Sequencing Protocol
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Before sequencing, the PCR products were purified using a Qiagen PCR purification kit (Qiagen) following manufacturer’s instruction. The cycle sequencing PCR was then performed by BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) applying manufacturer’s instructions. The thermal cycling profile comprised (i) initial denaturation at 94°C for 1 minute, (ii) 25 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 5 seconds and extension at 60°C for 4 minutes, and (iii) final extension at 60°C for 10 minutes. After cycle sequencing PCR, the products were purified using BigDye XTerminator® Purification Kit (Applied Biosystems). Then, sequencing of the purified cycle sequencing products was executed on the ABI PRISM 310 automated sequencer (Applied Biosystems, USA) [7 (link)].
3
Cycle Sequencing Protocol for DNA Characterization
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Prior to sequencing, PCR product was purified using a Qiagen PCR purification kit (Qiagen) according to manufacturer's instruction. The cycle sequencing PCR was then conducted using a BigDye Chain Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, USA) following manufacturer's instructions. The thermal cycling profile included (a) initial denaturation at 94°C for 1 minute, (b) 25 cycles of denaturation at 94°C for 10 seconds, annealing at 58°C for 5 seconds and extension at 60°C for 4 minutes, and (c) final extension at 60°C for 10 minutes. Following completion of cycle sequencing PCR, purification of the product was performed using a BigDye XTerminator® Purification Kit (Applied Biosystems). Finally, sequencing of the purified cycle sequencing product was performed on the ABI PRISM 310 automated sequencer (Applied Biosystems, USA).
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