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Optimization Of Primary Annealing Temperature With Bigdye Reagent In Sequencing Reaction
[Full Text]
AUTHOR(S)
Harumi Yuniarti, Astri Rinanti, Bambang Cholis S.
KEYWORDS
Bigdye Reagent, Primery Sequencing, PCR, Sequencing, Annealing Temperature, Electropherogram,
ABSTRACT
In this research, the annealing temperature was varied to determine the appropriate template sequence. The amplification process used the polymerase chain reaction (PCR) method in the Primary template, to separate the double DNA into a single chain. Furthermore, the cycling duration were compared with the pGEM_Standard. In this research, the process was heated for some time, and the temperature decreased to obtain an appropriate result. Bigdye-pGEM reagents were used to stick the separated molds to become single chains. Also, a primer pair with a large melting temperature difference tends to cause a reduction in the amplification process. The primary sequencing of M13 at 500C produced a well-readable amplicon on the observed electropherogram using ABI Prism 310 sequencer. The results shows that the sequencing test with the addition of bigdye reagent volume (without buffer) at 1x concentration and 25 times cycling duration formed high and clear peaks around 600bp. Shorter sequences occurred at lower concentrations, with the reagent used to determine the exact annealing temperature and how optimal the reagent brings up the sequence length that appears on the electropherogram.
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