Research Article http:sciencetechindonesia.com Science & Technology Indonesia p-ISSN: 2580-4405 e-ISSN: 2580-4391 Sci. Technol. Indonesia 3 (2018) 26-29 © 2018 The Authors. Production and hosting by ARTS Publishing in association with Indonesian Science and Technology Society. This is an open access article under the CC-BY-NC-SA license. Article History: Received 22 November 2017; revised 16 January 2018; accepted 16 January 2018 http://doi.org/10.26554/sti.2018.3.1.26-29 Comparison Of CTAB Method And Wizard Genomic Dna Purification System Kit From Promega On DNA Isolation Of Local Varieties Of Rice Of South Su- matera Laila Hanum1*, Yuanita Windusari1, Arum Setiawan1, Muharni1, Fikri Adriansyah1, Amin Ali Mubarok1 1Departement of Biology, Mathematic and Science Faculty, Sriwijaya University *Corresponding Author e-mail: lailahanum@ymail.com ABSTRACT Research in the molecular field requires DNA with a high degree of purity. Local rice varieties of South Sumatra have different leaf texture so that in isolation process to obtain DNA with high purity level required the right method. This study aims to compare the quality and quantity of purity of local rice insulation of local rice varieties of South Sumatra with different methods of CTAB and Wizard Genomic DNA Purification System Kit from Promega. The research was conducted from August 2015 to December 2015 at the Laboratory of Microbiology and Genetics and Biotechnology Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Sriwijaya University. The method used is DNA isolation method Wizard Genomic DNA Purification System Kit from promega and CTAB. Based on research that has been done result of DNA visualization showed that DNA isolate CTAB method still have smears while DNA isolate method of Wizard Genomic DNA Purification System Kit from promega not. The average DNA isolate concentration of Wizard Genomic DNA Purification System Kit method is A260 / 280 = 1.853 μg / ml. The average DNA isolate concentration of CTAB method is A260 / 280 = 1,705 μg / ml. Isolation of Rice DNA of local variety of South Sumatera using DNA method of Genomic DNA Purification System Kit from promega has higher quality and quantity compared to CTAB method. Keywords: CTAB, Local Rice Varieties of South Sumatra, Promega 1. INTRODUCTION Local rice varieties of South Sumatra have superior properties that are not owned by superior rice varieties. These properties are stored in genes. This property is an important source of ger- mplasm to be maintained. In addition, genetic information can be used as a base material in the process of plant breeding. The genetic information of a plant variety can be known by the PCR technique. DNA isolation was the first step in molecular analysis of PCR (Restu et al., 2012). The purity of target DNA is very important in the PCR process, since impurities of DNA sus- pension can affect the amplification reaction and may inhibit the action of DNA polymerase enzymes. Selection of the target to be amplified should take into ac- count the genetic stability of the targeted region or nucleotide sequence (Fatchiyah et al., 2011).This study aims to compare the results of isolation of local rice varieties of rice with different methods of CTAB method and Wizard Genomic DNA Purifica- tion System Kit from Promega. So as to determine which type of method is appropriate for DNA isolation. High quality and high quantity of DNA and free of contaminants can be used for other studies using PCR. 2. EXPERIMENTAL SECTION 2.1. Materials and Equipments The tool used in this research is stationery, aluminum foil, ana- lytical scale, ice box, glove, mortar, pestle, mikropipet, yellow tip, microsentrifus, microtube size 0.2 ml and 1.5 ml, hot plate, spec- trophotometer, thermo-cycler, waterbath, and Wizard Genomic DNA Purification System Kit from Promega and CTAB. Materials needed are for DNA isolation is the local rice leaf of local varieties of South Sumatra (Table 1), ice, ethanol 70%, ethanol 96-100%. 2.2. DNA Isolation method Wizard Genomic DNA Puri- fication System Kit from Promega The leaves are frozen in the freezer as much as 0.1 gr. Then finely crushed while added Nuclei Lisis Solution (NLS) 600 μL then put in eppendof tube size 0,5 μL and in vortex for 3 second. Incubated Hanum et al. 2018 / Science & technology Indonesia 3 (1) 2017: 26-29 27 in water bath for 15 minutes at 650C. Added 200 μL PPS then divortex for 20 seconds. Then centrifuged at 13,000 rpm for 3 minutes. Supernattan is transferred into 600 μL isopropanol (0.6) volume. Mix the solution until you see the white thread of DNA. Then centrifuged at 13,000 rpm for 1 minute. Supernattan was removed and 600 μL of ethanol was added 70%. Wash the DNA by centrifuging at 13,000 rpm for 1 minute. Discard the supernat- tan and then dry the DNA for 15 minutes. Finally added 100 μL of DRS DNA and store DNA at a temperature of 2-80 C 2.3.CTAB Conducted by method of Doyle and Doyle (1987) in Restu et al. (2012) modified. A total of 200 mg of leafless rice leaves plus 0,02 g of PVP were crushed until smooth (flour). The crushing results were then transferred into a 1.5 ml eppendorf tube, plus 0.5 ml of CTAB extraction buffer (1.4 M NaCl, 2% CTAB, 50 mM EDTA, 1 M Tris-HCl pH 8.0 and 0, 2% ß-mercaptoetanol). The process of lysis of the cell wall is done by incubating the tube containing the leaf sample into the temperature waterbath of 65oC for 60 minutes. The tube is lifted from the waterbath and allowed for sev- eral minutes until the sample temperature in the tube decreases. Subsequently added chloroform: isoamilalcohol (CIA 24: 1) 500 μl. The tube is shaken using a vortex, then centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred into a bar tub and then cold isopropanol was added as much as 1 volume, slowly flipped through until the DNA thread was visible. The sam- ple was then allowed to settle overnight in the refrigerator at 4 ° C. After being deposited overnight, the sample was then centrifuged for 15 minutes at 10,000 rpm. The pellets of DNA formed at the bottom of the tube are then dried. After that 100 μl H2O was added and stored in refrigerator (-40 C). 2.4. Electrophoresis Weighed 0.5 gr of agarosa and then put into a erlenmeyer flask containing 50 ml of TAE while shaking to dissolve. Insert a mag- netic stirel into the flask and heat on the hotplate until cooked. Poured into a well comb which amounts to 8 wells on the first comb and 16 wells on the second comb slowly so as not to form bubbles. Let stand until the gel or gel for ± 30 minutes and after cool comb then lifted. The 10 μL DNA samples were pipetted and then inserted into the wells contained in the gel using a mi- crophone. Added 0.7 μL loading dye in the well. The gel is then fed into an electrophoresis tank that has been filled with a TAE buffer. Electrode connected with power supply at 100 volt voltage with ampere 400 ohm for 30 minutes then running done for 30 minutes. After running is done then pour 200 ml of TAE and add a drop of EtBr into aluminum foil coated container. Inserted aga- rose gel into the container and then soaked for 15 minutes. 2.5. Electrophoresis Visualization of Electrophoresis Results Visualization of the results running on the gel is done by using a UV transilluminator and the results are photographed as docu- mentation. 2.6. Quantification and Quality of Isolated DNA Result Prior to use in PCR reactions, DNA quantification was done to determine the purity and concentration of DNA. Quantification is done using GeneQuant (Life Science, Ltd., UK). A 2 μl isolated DNA sample was fed into a quartet containing 1998 μl sterile aq- uabides and mixed homogeneously. The sample then calculated its concentration at wavelength (λ) 260 and 280 nm. Comparison of concentrations at each wavelength is used as a benchmark of DNA purity. DNA purity is determined on the basis of the ratio of A260 / 280 = 1.8-2 [8]. DNA quality testing can also be performed with agarose gel electrophoresis on genomic DNA prior to PCR. DNA quality is good when the DNA bands appear clear, thick, there is only one band and no shadow under the band (smear) when com- pared to marker (Sambrook et al., 1989) 3. RESULTS AND DISCUSSION 3.1. CTAB Based on the results of electrophoresis (Figure 1) and reinforced with the result of quantity test and DNA quality test (Table 2) shows the isolated DNA with CTAB method can still be used for the analysis of genetic variation by RAPD method. Although its purity is not in accordance with the specified standards. This is based on the ratio of concentration at each wavelength used as a benchmark of DNA purity. DNA purity was determined on the basis of the ratio of A260 / 280 = 1.8-2 (Sambrook et al., 1989). The impure DNA of the mold still contains RNA which is marked by the presence of Smears on the visualization result of the DNA band. This will disrupt the primary attachment of the site and will inhibit the activity of DNA polymerase enzymes. This enzyme serves to polymerize the DNA while the molded DNA that Table 1. List of Local Varieties of Rice Soyth Sumatra No. Varieties Code 1 Padi Pegagan PP 2 Padi Dayang Rindu DRMK 3 Padi Dayang Kuning DKMK 4 Padi Seluang SMK 5 Padi Panak/Pendek PMK 6 Padi Pamulan/Empat Bulan PEBMK 7 Padi Talang TB 8 Padi Sanapi SB 9 Padi Ketan Itam KIB 10 Padi Ketan Putih KPB 11 Padi Ketan Abang/Ketan Merah KAB 12 Padi Dayang Telasih DTLLG 13 Padi Hitam HLLG 14 Padi Pulut/Meto Tomok PLLG1 15 Padi Putih PLLG2 16 Padi Pengagat PLLG3 17 Padi Dayang Rindu DRLLG 18 Padi Pulut PM2SI 19 Padi Panjang PM2S2 20 Padi Jambat Thehas JTM2S 21 Padi Beram BM2S 22 Padi Stik SK Hanum et al. 2018 / Science & technology Indonesia 3 (1) 2017: 26-29 28 many have fragmentation can eliminate the primary attachment site. Based on the results of electrophoresis (Figure 2) and rein- forced by the result of quantity test and DNA quality test (Table 3) shows the isolated DNA with Wizard Genomic DNA Purificati- on System Kit method from Promega can be used for analysis of genetic variation by RAPD method. This is based on the ratio of concentration at each wavelength used as a benchmark of DNA purity. DNA purity was determined based on the ratio of A260 / 280 = 1.8-2 (Sambrook et al., 1989). 3.2. DNA Purification System Kit from Promega One of the advantages of the diversity analysis utilizing PCR technology is that the quantity of DNA required is small (Arif et al.,2010). The molded DNA concentration used ranges from 1.5 to 25 ng in each PCR reaction. Determination of the concentra- tion of molded DNA greatly affects the attachment of primers to the molded DNA. High DNA mold concentrations will result in poor DNA amplification because the primers used are difficult to stick to. Conversely, if the concentration of the molded DNA is too low, there will be a competition where the primer attaches to the mold causing one of the fragments to be amplified in large quantities while the other fragment is slightly (Pharmawati, 2009). The optimization of isolation and purification technique of DNA on the betel nut plant (Reutalis trisperma (Blanco) obtained DNA quantity ranged from 68.80-5.031.39 ng / μl with DNA purity in the range of 1,8-1,9 with electrophoresis result showed intact DNA marked with absence of DNA smears) (Syafarudin and Santoso, 2011). The ratio is too low (<1.8) contains too much RNA, while the ratio is too high (> 2.2) indicates the presence of many protein contaminants in the DNA solution. DNA purity of the template (DNA template) greatly affects the intensity of amplified DNA bands. DNA molds containing compounds such as polysaccharides and phenolic compounds will result in a faint or unclear amplification DNA band (Sambrook et al., 1989). Genetic markers (RAPD) are very sensitive to reaction conditions and DNA purity of the mold (Young et al., 2000). CONCLUSION Isolation of Rice DNA of local variety of South Sumatera using DNA method of Genomic DNA Purification System Kit from Table 2. Quantification of DNA CTAB Method No Sample Quantity 1 Padi Pegagan 1,74 2 Padi Dayang Rindu 1,73 3 Padi Dayang Kuning 1,92 4 Padi Seluang 1,72 5 Padi Panak/Pendek 1,68 6 Padi Pamulan/4 bulan 1,65 7 Padi Talang 1,75 8 Padi Sanapi 1,68 9 Padi Ketan Itam 1,66 10 Padi Ketan Putih 1,71 11 Padi Ketan Abang 1,67 12 Padi Meto Tomok 1,68 13 Padi Dayang Telasih 1,71 14 Padi Pengagat 1,60 15 Padi Hitam 1,69 16 Padi Putih 1,57 17 Padi Pulut 1,72 18 Padi Dayang Rindu 1,67 19 Padi Panjang 1,76 20 Padi Jambat Thehas 1,74 21 Padi Beram 1,72 22 Padi Stik 1,74 Table 3. Quatification of DNA Using Promega No Sample Quantity 1 Padi Pegagan 1.806 2 Padi Dayang Rindu 1.86 3 Padi Dayang Kuning 1.839 4 Padi Seluang 1.889 5 Padi Panak/Pendek 1.863 6 Padi Pamulan/4 bulan 1.802 7 Padi Talang 1,820 8 Padi Sanapi 1.985 9 Padi Ketan Itam 1.828 10 Padi Ketan Putih 1.808 11 Padi Ketan Abang 1.804 12 Padi Meto Tomok 1.848 13 Padi Dayang Telasih 1,857 14 Padi Pengagat 1.897 15 Padi Hitam 1.877 16 Padi Putih 1.893 17 Padi Pulut 1.852 18 Padi Dayang Rindu 1.815 19 Padi Panjang 1,881 20 Padi Jambat Thehas 1,898 21 Padi Beram 1.843 22 Padi Stik 1.804 Figure 2. Result of DNA Band Visualization Wizard Genomic DNA Purification System Kit Promega. Figure 1. Result of DNA Band Visualization CTAB Method Hanum et al. 2018 / Science & technology Indonesia 3 (1) 2017: 26-29 29 promega has higher quality and quantity compared with CTAB method isolate. REFERENCES Arif, I.A., Bakir, M.A., Khan, H.A., Al Farhan, A.H., Al Ho- maidan, A.A., Bahkali, A.H., Al Sadoon, M. and Shobrak, M. 2010. Application of RAPD for molecular characterization of plant species of medicinal value from an arid environment. Genet. Mol. Res. 9(4): 2191-9198. Fatchiyah, A.E.L., Widyarti, S., dan Rahayu, S. 2011. Biologi Mol- ekuler: Prinsip Dasar Analisis. Jakarta: Erlangga xxiv + 191hlm. Karmana, Wayan, I. 2009 Adopsi Tanaman Transgenik dan Be- berapa Aspek Perkembangannya. GeneC Swara. 3(2): 12-21. Pharmawati M. 2009. Optimalisasi Ekstraksi DNA dan PCR- RAPD pada Grevillea spp. (Proteaceae). Jurnal Biologi, 8(1) : 12 -16. Poerba, Yuyu., S., Martanti, D. 2008. Keanekaragaman Genetik berdasarkan Marka Random Amplified Polymorphic DNA pada Amorphophallus muelleri Blume di Jawa. Biodiversitas. 9(4): 245-249. Restu M, Mukrimin, dan Gusmiaty. 2012. Optimalisasi Teknik Ekstraksi dan Isolasi DNA Tanaman Suren (Toona Sureni Merr.) untuk Analisis Keragaman Genetik berdasarkan Random Ampli- fied Polymorphic DNA (RAPD). Jurnal Natur Indonesia, 14(2): 138- 142. Sambrook, J., Fritsh, E.F. and Maniatis T. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press. New York. Young Et., Sloan JS., Van Riper K. 2000. Trinucleotide Repeats are Clustered in Regulatory Genes in Saccharomyches cerevisiae. Genetics. 154(3): 1053-68.