Maggy T. Suhartono
Departemen Ilmu dan Teknologi Pangan, Fakultas Teknologi Pertanian, Institut Pertanian Bogor, Bogor

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MORPHOLOGYCAL AND MOLECULAR PARTIAL HISTONE-H3 CHARACTERIZATION OF BINTAN SEA SNAIL GONGGONG (STROMBUS SP.) AS A SPECIES VALIDATION Viruly, Lily; Andarwulan, Nuri; Suhartono, Maggy T.; Nurilmala, Mala
HAYATI Journal of Biosciences Vol. 26 No. 2 (2019): April 2019
Publisher : Bogor Agricultural University, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (658.468 KB) | DOI: 10.4308/hjb.26.2.%x

Abstract

Sea snail gonggong is an icon of Tanjungpinang-Riau Islands Province. It is a favorite seafood item in Riau Islands Province, and is high economic value but not known widely yet. Until now, sea snail gonggong has been highly exploited but the research on this snail is very limited. The aim of this study was to morphology and molecular characterization of Bintan gonggong snail (Strombus sp.) as a species validation. Bintan gonggong snail included thick-shelled gonggong and thin-shelled gonggong. Morphology identifcation of species Bintan gonggong snail was based on morphometric variability. Molecular identifcation used partial Histone-H3, MEGA version 6.06, and bioinformatics analysis. The result showed that the morphological identifcation of thick-shelled and thin-shelled gonggong based on shell width, the lip thickness, and total weight signifcantly di?erent, but other variables (i.e shell length, shell depth, aperture length, and gonggong weight) were not signifcantly di?erent (p<0.05). Resulted of a molecular identifcation with phylogenetic analysis that the thin-shelled and thick-shelled Bintan gonggong snails were 1 species and a genetic distance of 1%. They were not species Strombus canarium, Strombus vitatus, and Strombus epidromis. Bintan gonggong snails were Strombus turturella (Leavistrombus turturella). DNA sequences of Bintan gonggong have been registered in Gen-Bank with registration numbers MH348131 (thinshelled gonggong) and MH348132 (thick-shelled gonggong).
AKTIVITAS IGY DAN IGG ANTITETANUS SETELAH PERLAKUAN PADA BERBAGAI PH, SUHU DAN ENZIM PROTEOLITIK Suartini, I Gusti Ayu Agung; Teguh Wibawan, I Wayan; Suhartono, Maggy T.; -, Supar; Suarta, I Nyoman
Jurnal Veteriner Vol 8 No 4 (2007)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

A study was carried out to find out an alternative method of producing antitetanus antibody (IgY) in chicken and to evaluate its activity at different levels of pH, temperatures and proteolytic enzymes. Antitetanus IgY was produced by immunization of chickens with tetanus toxoid, three times weekly at gradual doses of 100, 200, and 300 Lf, respectively. Serum samples were collected 4 weeks following the last immunization. IgY was purified by ammonium sulfat precipitation and gel filtration chromatography (Sephadex G. 120).The purified IgY was then treated at different levels of temperatures and pH as well as proteolytic enzymes. Commercial antitetanus IgG was used as control. The activities of treated IgY and IgG were tested by enzyme linked immunosorbent assay (ELISA). IgY and IgG activities were significantly reduced at 80ºC and completely destroyed at 90ºC. Treatment with pepsin significantly reduced IgY and IgG whereas trypsin slightly reduced IgY activities and has no effect on IgG activities. IgY and IgG activities were reduced significantly at pH < 3 and and only sightly reduced at pH>10. It is evident that heating at >90oC, pH at <3 and treatment with pepsin significantly reduced IgY activities and it appears that IgG was more resistent to the efect of temperatures, pH and proteolytic enzymes
PROTEIN HISTON PADA SIPUT GONGGONG BINTAN STROMBUS SP. SEBAGAI KANDIDAT PANGAN FUNGSIONAL Viruly, Lily; Andarwulan, Nuri; Suhartono, Maggy T.; Nurilmala, Mala
Jurnal Ilmu dan Teknologi Kelautan Tropis Vol. 11 No. 1 (2019): Jurnal Ilmu dan Teknologi Kelautan Tropis
Publisher : Department of Marine Science and Technology, Faculty of Fisheries and Marine Science, IPB University

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1114.559 KB) | DOI: 10.29244/jitkt.v11i1.22299

Abstract

ABSTRAKGonggong termasuk sejenis siput laut, biota endemik yang banyak hidup di pantai Pulau Bintan dan sekitarnya di Provinsi Kepulauan Riau. Gonggong merupakan ikon kota Tanjungpinang, Provinsi Kepulauan Riau. Sampai saat ini, penelitian gonggong masih sangat sedikit padahal siput ini merupakan spesies yang sangat potensial. Tujuan penelitian ini adalah untuk mengkarakterisasi protein histon dari siput gonggong Strombus sp. asal Bintan sebagai kandidat pangan fungsional. Karakterisasi profil protein menggunakan SDS-PAGE. Kadar protein pada gonggong dianalisis dengan menggunakan metode Bradford. Gonggong rebus bercangkang tebal diekstraksi dengan metode maserasi menggunakan pelarut etanol PA 95% dan uji aktivitas antimikroba menggunakan metode sumur. Asam amino dianalisis menggunakan HPLC. Hasil karakterisasi profil protein pada daging gonggong menunjukkan bahwa gonggong bercangkang tipis dan gonggong bercangkang tebal memiliki pita profil protein yang sama pada berat molekul 11-37 kDa, sedangkan profil protein pada lendir gonggong bercangkang tebal dan tipis memiliki pita protein yang sama pada berat molekul 37 kDa. Jenis protein pada spesies gonggong Bintan diprediksi merupakan protein histon karena hasil amplifikasi menggunakan primer protein histon H2A dan H2B didapatkan gen target pada 75 bp dan uji antimikroba pada bakteri S. aureus dan E. coli memiliki nilai DDH sebesar 25,65±0,02 mm dan 14,45±0,13 mm, sehingga diduga bahwa gonggong Bintan berpotensi sebagai kandidat pangan fungsional khas Kepulauan Riau. ABSTRACTGonggong is one of the sea snails, endemic species living on coastal waters of Bintan Island and surrounding islands of the Riau Islands Province. Sea snail gonggong is an icon of Tanjungpinang-Riau Islands Province. Until now, research this snail is the least, whereas it is potential species. The purpose of this study was to characterize histone protein from Bintan gonggong snail Strombus sp. as functional food candidate. Protein profiling used SDS-PAGE. Protein contents were analyzed by Bradford method. Boiled thick shelled gonggong were extracted by maseration method using ethanol PA 95% and antimicrobial activity tes using well method. Amino acid analized with HPLC. The result of characterization on protein profiles in meat gonggong showed that the thin-shelled and thick-shelled gonggong had the same band as protein profiles by 11-37 kDa and protein profiles in mucus gonggong were found the same band as protein profiles of 37 kDa. The type of protein in spesies Bintan gonggong had been predicted a histone protein because DNA identification using primer protein histone H2A and H2B had gen target of 75 bp. Antimicrobial activity test on S. aureus and E. coli bacteria had value DDH of 25.65±0.02 mm and 14.45±0.13 mm. In fact, gonggong snail was potentially as antimicrobial peptide, so it will make local functional food candidate from Riau Islands Province.
AKTIVITAS ANTIBAKTERI FRAKSI-FRAKSI EKSTRAK SIRIH HIJAU (PIPER BETLE LINN) TERHADAP PATOGEN PANGAN [ANTIBACTERIAL ACTIVITY OF FRACTIONATED GREEN SIRIH (PIPER BETLE LINN) EXTRACT AGAINST FOOD PATHOGENIC BACTERIA] Suliantari, .; Jenie, Betty S. L.; Suhartono, Maggy T.
Jurnal Teknologi dan Industri Pangan Vol. 23 No. 2 (2012): Jurnal Teknologi dan Industri Pangan
Publisher : Departemen Ilmu dan Teknologi Pangan, IPB Indonesia bekerjasama dengan PATPI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (273.882 KB) | DOI: 10.6066/jtip.2012.23.2.217

Abstract

Fractionation of green sirih (Piper betle Linn) extract by chromatography colom using the mixture of several solvents i.e. chloroform, ethanol and acetic acid (4:1:1) resulted in 17 fractions. All fractions showed antibacterial activities but only 2 fractions (fraction 3 and fraction 4) showed the highest inhibition towards the six tested bacteria Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. Among the tested bacteria, all fractions of green sirih extracts showed the most effective inhibition against, Salmonella Typhimurium with inhibition zone diameters ranging from 10 mm to 26 mm. Identification using GC-MS found that fraction 3 and fraction 4 contained chavicol; dodecanoic acid, myristic, palmitic and oleic acid.  
FRAKSIONASI DAN PENENTUAN PROFIL PROTEIN BUNGKIL KELAPA DENGAN SDS-PAGE [FRACTIONATION AND PROFILING OF COPRA MEAL’S PROTEIN BY USING SDS-PAGE] Wibowo, Agus Danang; Suhartono, Maggy T.; Siagian, Patuan L. P.
Jurnal Teknologi dan Industri Pangan Vol. 23 No. 1 (2012): Jurnal Teknologi dan Industri Pangan
Publisher : Departemen Ilmu dan Teknologi Pangan, IPB Indonesia bekerjasama dengan PATPI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (300.062 KB)

Abstract

The aim of this research was to extract proteins from the copra meal, base on their solubility and analysis of the protein profiles by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) This research was conducted in two stages, protein fractionation and molecular weight estimation using SDS PAGE. The fractionation was conducted by non-enzymatic treatment and enzymatic (mannanase) treatment. Kjeldahl analysis showed that the protein content of copra meal was 18.52% of dry basis. Protein fractionation could separate the copra meal?s proteins based on their solubility. Albumin was solubilized by water, globulin by NaCl 5%, glutelin by NaOH 0.02M, and prolamin by ethanol 70%. Fractionation using non-enzymatic treatment resulted in 64% of albumin, 39.25% of globulin, 15.27% of glutelin, and 38.84% of prolamin fractions. On the other hand, fractionation using enzymatic treatment resulted different protein profile, giving 28.17%, 39.44%, 10.91%, and 21.48% of the respective proteins. Characterization using SDS PAGE showed that the protein profile from the non-enzymatic was different from that of the enzymatic treatment. In conclusion, we found that the proteins extracted from the copra meal with and without mannanase showed variation in the range of molecular weights: 45.1-66,0 kDa and 66.1-97.4 kDa, respectively.  
PEMURNIAN DAN KARAKTERISASI INHIBITOR PROTEASE DARI CHROMOHALOBACTER SP. 6A3, BAKTERI YANG BERASOSIASI DENGAN SPONS XETOSPONGIA TESTUDINARIA [PURIFICATION AND CHARACTERIZATION OF PROTEASE INHIBITOR FROM CHROMOHALOBACTER SP. 6A3, BACTERIA-ASSOCIATED WITH S Nurhayati, Tati; Suhartono, Maggy T.; Nuraida, Lilis; Poerwanto, Sri Budiarti
Jurnal Teknologi dan Industri Pangan Vol. 21 No. 2 (2010): Jurnal Teknologi dan Industri Pangan
Publisher : Departemen Ilmu dan Teknologi Pangan, IPB Indonesia bekerjasama dengan PATPI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (464.732 KB)

Abstract

 Various sponges has been reported to produce protease inhibitor which could inhibit protease activity of pathogenic bacteria. The previous research showed that bacteria-associated with sponge could produce bioactive compound similar to their host, including protease inhibitor. The purposes of this research were to purify protease inhibitor from Chromohalobacter sp. 6A3 and to study the characteristics of the protease inhibitor. The result showed that the protein can be extracted by 30 % (v/v) acetone, purified by gel filtration (Sephadex G-75) and finally, purified by anion exchanger (Sephadex A-50). The molecular weight of the purified protease inhibitor after gel filtration was estimated as 21,31 kDa and 17,05 kDa, but anion exchanger gave protein with estimated molecular weight of 21,31 kDa The optimum temperature and pH were 30 oC and 5 respectively. The protease inhibitor could resist heating at 30 oC for 10 minutes. Incubation of the inhibitor at 30 oC, pH 5, still retained its activity until 3 hours. The purified enzyme inhibitor was also still active after incubated at pH from 5 to 6 for 1 hour. The most susceptible substrate (enzyme) for the inhibitor was protease from P. aeruginosa. The protease inhibitor was inhibited by metal ions except Na+ 1mM. Activity of the inhibitor increased twofold by SDS 5 mM. IC 50 of the protease inhibitor was 3.48 nM. The protease inhibitor inhibited the enzyme uncompetitively.
Partial Purification and Characterization of Chitin Deacetylase Produced by Bacillus thermoleovorans LW-4-11 Toharisman, Aris; Suhartono, Maggy T.
JURNAL BIOLOGI INDONESIA Vol 4, No 4 (2007): JURNAL BIOLOGI INDONESIA
Publisher : Perhimpunan Biologi Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/jbi.v4i4.3249

Abstract

ABSTRACTPurifikasi Parsial dan Karakterisasi Enzim Kitin Deasetilase yang Dihasilkan Bacillus thermoleovorans LW-4-11. Kitin deasetilase yang dihasilkan dari bakteri Bacillus thermoleovorans LW-4-11 telah dimurnikan menggunakan amonium sulfat 70% diikuti dengan perlakuan panas (suhu 70°C selama 1 jam). Kemurnian enzim yangdipisahkan dari medium fermentasi meningkat 4.28 kali dengan aktivitas spesifik sekitar 43.7 mUImg. Enzim memiliki suhu dan pH optimum masing-masing 80°C dan 6.0 dalam substrat 0-hydroxyethylated chitin (glycol chitin). Kitindeasetilase ini relatif tahan panas dengan waktu-paruh sekitar 30 menit pada suhu 80 "C. Enzim dihambat oleh ion Li, ZnZ+, MnZ+, Co2 and Ni pada konsentrasi 1 mM, tetapi diaktifkan oleh EDTA 1 mM.Kata kunci: purifikasi parsial, karakterisasi, kitin deasetilase, B. thermoleovorans LW-4-11
Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen NURHAIMI-HARIS, .; ASWIDINNOOR, Hajrial; SUWANTO, Antonius; SUHARTONO, Maggy T.; TORUAN-MATHIUS, Nurita; PURWANTARA, Agus
E-Journal Menara Perkebunan Vol 73, No 2: Desember 2005
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v73i2.156

Abstract

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.
APLIKASI MUTAN BERFLUORESENS UNTUK MEMPELAJARI KETAHANAN HIDUP, KOLONISASI DAN PENETRASI ISOLAT CRONOBACTER SAKAZAKII SELAMA PENGERINGAN JAGUNG [USE OF GFP MUTANT TO STUDY THE SURVIVAL, COLONIZATION AND PENETRATION OF CRONOBACTER SAKAZAKII ISOLATES DURI Nurjanah, Siti; Suhartono, Maggy T.; Hariyadi, Ratih Dewanti-; Estuningsih, Sri
Jurnal Teknologi dan Industri Pangan Vol. 24 No. 2 (2013): Jurnal Teknologi dan Industri Pangan
Publisher : Departemen Ilmu dan Teknologi Pangan, IPB Indonesia bekerjasama dengan PATPI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (616.174 KB) | DOI: 10.6066/jtip.2013.24.2.184

Abstract

Cronobacter sakazakii is a Gram-negative emerging pathogen regarded as causative agent of meningitis and necrotizing enterocolitis in certain groups of infants. In the previous research, thirty-two local isolates of C. sakazakii were obtained from various dried food products such as from corn starch, suggesting that they are able to survive drying. Some of the isolates were toxic. Green Fluorescent Protein (GFP) have been inserted to C. sakazakii and used as a marker for selective enumeration due to the ability of this protein to fluoresce under UV and to tolerate in ampicillin containing media. The objective of this study was to evaluate the survival, colonization and penetration of two isolates of C. sakazakii from dried food product during maize drying. The maize was challenged with mutants at a concentration of 105-106 CFU/g before drying. Maize drying was performed at temperature of 40ºC, 45ºC and 50ºC for 4, 6 and 8 days until the moisture content reached 14%. The totals of resistant drying mutants were counted every day onto ampicillin containing media by observing under UV light. The survival rate of C. sakazakii during drying was determined by the slope of linier regression from C. sakazakii survival curve. Isolates of FWHd16, the toxic strain of C. sakazakii, were more resistant to heat treatments in comparison to isolates of YRt2a, or the non toxic strain of C. sakazakii. Following fluorescence and scanning electron microscope observation, it is concluded that both isolates were colonizing on maize surface. These mutants were able to penetrate to the inner side of the grain by entering injured surface or pores at the tip cap of maize.
Konstruksi pustaka cDNA dari daun klon karet AVROS 2037 yang diinfeksi patogen Corynespora cassiicola Construction of a cDNA library from leaf of AVROS 2037 rubber clone infected by Corynespora cassiicola pathogen NURHAIMI-HARIS, .; ASWIDINNOOR, Hajrial; SUWANTO, Antonius; SUHARTONO, Maggy T.; TORUAN-MATHIUS, Nurita; PURWANTARA, Agus
E-Journal Menara Perkebunan Vol 73, No 2: Desember 2005
Publisher : INDONESIAN RESEARCH INSTITUTE FOR BIOTECHNOLOGY AND BIOINDUSTRY

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.22302/iribb.jur.mp.v73i2.156

Abstract

SummaryConstruction of cDNA library derived fromtranscripts made under certain condition is animportant first step to understand diseaseresistant mechanisms. To identify rubber genesor transcripts involved in defense responsetoward Corynespora cassiicola, cDNA librarywas constructed using rubber clone AVROS2037, one of resistant clone to this pathogen.cDNA library was constructed based on thestrategy of leaves infection using conidia, withthe assumption that transcript expression relatedto defense response would be induced bypathogen infection. RNA was isolated from leavesthree days after inoculation with conidia ofC. cassiicola. Steps involved in the cDNA libraryconstruction were RNA isolation, mRNApurification, cDNA synthesis, vector modifcation,cDNA insert ligation, plasmid transformation andclone verifications. Each gram of leaf producedapproximately 300  g RNA, and 0.25% of themwas mRNA. The mRNA was used to synthesizedcDNA. Ligation of cDNA and modified vectorwas facilitated by restriction enzyme SfiI. Theconstructs were transformed into the E. coliDH5 competent cells. A total of 8000 colonieswere produced. Random examination of 270colonies showed that approximately 93% of thesecolonies carried plasmid vector with DNA insertsize of 200 – 2000 bp, with average size of 500 –800 bp. cDNA library construction of rubberleaves from AVROS 2037 clone as well as somenecessary modification steps are presented in thispaper.RingkasanKonstruksi pustaka cDNA yang me-ngandung transkrip yang diekspresikan dalamkondisi tertentu merupakan tahap awal yangsangat penting dalam berbagai studi biologi.Untuk mengidentifikasi gen karet atau transkripyang berperan dalam respons pertahanan tanamankaret terhadap Corynespora cassiicola, pustakacDNA dibuat dengan menggunakan daun klonAVROS 2037 yang merupakan salah satu klonresisten terhadap patogen tersebut. PustakacDNA dibuat berdasarkan strategi menginfeksidaun dengan konidia C. cassiicola denganpertimbangan bahwa ekspresi transkrip yangberperan dalam respons pertahanan akandiinduksi oleh adanya infeksi patogen. Dengandemikian pustaka cDNA yang dibuat diharapkanmengandung gen atau bagian gen yang ber-hubungan dengan respons pertahanan. RNAdiisolasi dari daun setelah daun diinokulasiselama tiga hari dengan konidia C. cassiicola.Beberapa tahapan telah dilakukan, dimulaidengan isolasi RNA, pemurnian mRNA, sintesiscDNA, modifikasi vektor kloning, ligasi fragmencDNA utas ganda dengan vektor kloning sertatransformasi hasil ligasi ke bakteri Escherichiacoli DH5 kompeten. Dari setiap gram jaringandaun berhasil diisolasi RNA sekitar 300 g, dandari jumlah tersebut sekitar 0,25% mRNA dapatdiisolasi. mRNA yang diisolasi digunakan untuksintesis cDNA. cDNA dipotong dengan enzimrestriksi SfiI dan diligasi ke vektor plasmid yangdimodifikasi dengan menyisipkan situs enzimSfiI. cDNA-vektor rekombinan ditransformasi kedalam sel bakteri E. coli DH5 kompeten meng-gunakan metode standar. Transformasi konstrukini menghasilkan 8.000 koloni. Pengujian PCRterhadap 270 koloni yang dipilih secara acakmengindikasikan bahwa sekitar 93% kolonitersebut membawa cDNA sisipan dengan ukuranfragmen cDNA yang menyisip berkisar antara200 sampai 2000 bp. cDNA sisipan terbanyakterdapat pada ukuran antara 500 – 800 bp. Dalamtulisan ini dibahas tahap demi tahap proses yangdilakukan untuk membuat pustaka cDNA asaldaun karet klon AVROS 2037 serta beberapamodifikasi yang diperlukan.