INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 03, MARCH 2020 ISSN 2277-8616 1431 IJSTR©2020 www.ijstr.org Optimization Of Primary Annealing Temperature With Bigdye Reagent In Sequencing Reaction Harumi Yuniarti, Astri Rinanti, Bambang Cholis S. 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 50 0 C 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. Keywords---Bigdye Reagent, Primery Sequencing, PCR, Sequencing, Annealing Temperature, Electropherogram, ——————————  —————————— 1. INTRODUCTION THE Sanger method was used to analyze the DNA sequencing with polymerase used as an enzyme subunit to synthesize the presence of dNTP. This method was also used to distinguish dNTP from ddNTP with ABI prism-310 sequencer optimized by setting and determining the composition of the reagent using several processes. The appropriate temperature for the process, and cycling duration to determine the number of amplification cycles related to the reagent injection process was sorted by the capillary electrophoresis (CE) pipe [1], [2], [3]. In previous studies, DNA sequencing was carried out using the BigDye Terminator v3.1 reagent volume dilution method. The cycling duration of 25 times and at 50 0 C using a phydit program, obtained clear sequencing results with 99.74% similarity [4]. The temperature is estimated for the primer to stick to the DNA mold in a stable state. High annealing temperature makes the primary bond difficult, resulting in less efficient PCR products. Conversely, low temperature causes primary annealing to the DNA in an unspecified place [5]. For this reason, the study continued with variations in the concentration of the reagent, which was expected to determine how optimal the reagent is able to reveal the sequence. Therefore, the selection of reagent concentrations and the correct temperature setting allowed the determination of the sequence length needed in observations, in accordance with efficiency and cost 2. RESEARCH METHODOLOGYMATERIAL AND METHODS DNA fragments in the ACGT base sequence (Adhenine, Cytosine, Guanine, and Tymine) were used as templates (primers) and amplified using enzymes as well as materials complement the PCR reagent [6]. The amplification process using a PCR machine was carried out with a cycling duration of 25 times. The sequencing test was prepared for the composition of the reagent concentration using the pGEM-3Zf + reagent, by regulating the temperature at 96 ° C for 1 minute for the initial denaturation to occur. Each cycle was tested using a temperature of 49°C, 50°C and 51°C, then compared with pGEM_Standard. The next cycle sequencing was carried out in stages of successive heating, by denaturation at 96°C for 10 seconds, and 60°C extension for 4 minutes. Furthermore, it was stored at 4°C for purification, which was carried out using ethanol EDTA precipitation as a buffer running. In summary, the DNA sequencing process using the PCR (Polymerase Chain Reaction) was carried out in stages (Figure 1). The reagent concentration method was used to observe the sequences of the DNA process. The composition of the ingredients used are shown in Table 1, Sequencing test preparation In Table 1, the addition of 8 µL bigdye terminator v3.1 without buffer is considered the main reagent with concentration 1x, according to the initial procedure and a total volume of 20 µL in the capillary pipe (CE). Figure 1. Stages of DNA sequencing process --------------------------------------------------------------  Astri Rinanti, Department of Environmental Engineering, Faculty of Landscape Architecture and Environmental Technology, Trisakti University.  Bambang Cholis, Department of Industrial Engineering, Faculty of Industrial Technology, Trisakti University.  Corresponding Author is Harumi Yuniarti Department of Industrial Engineering, Faculty of Industrial Technology, Trisakti University, Jakarta, Indonesia. Corresponding Author E-mail: harumi@trisakti.ac.id