MATERIAL AND METHODSMaterial. Papay

MATERIAL AND METHODS

Material. Papaya samples were collected on the market. The fruit was peeled, the flesh was separated from the stones, cut into pieces and the slices of papaya flesh was dried in the oven at 37°C for 12 h to reduce the moisture content. Both the tissue were stored at –80°C until further processed.

The candied papaya was stored at room tem-perature until processed.
DNA isolation. From each sample two inde-pendent isolations were performed. Prior to DNA isolation, samples were homogenised in liquid nitrogen.

CTAB based DNA extraction. CTAB method (Cetyl trimethylammonium bromide) DNA isolota-tion was performed according ISO Standard (EN ISO 21571:2002). Total of 200 mg of powdered sample was used for DNA isolation. Extracted DNAs were eluted in 60 µl of 0.1 × TE buffer.

Wizard DNA extraction. DNAs were extracted and purified using the modified Wizard method. 200 mg of powdered sample was mixed with 860 µl of TNE buffer (5mM Tris-HCl, pH 8.0, 2mM EDTA, 150mM NaCl, 1% SDS). The mixture was vortexed. 100 µl of 5M guanidin hydrochloride and 40 µl of proteinase K hydrochloride were added to the solution, the mixture was vortexed and after that incubated at 58°C for 5 hours with occassionaly mix-ing. After incubation, the mixture was centrifuged for 10 min at 13 000 × g. 500 µl of supernatant was transferred into the new microtube and 10 µl of RNase was added to the sample. The sample was further incubated at 65°C for 10 minutes. DNA solu-tion was subsequently purified with Wizard DNA Clean-Up System (Promega, Madison, USA).

GeneSpin DNA Isolation Kit. DNA was extracted using GeneSpin DNA Extraction Kit (Eurofins, Freiburg, Germany) following the manufacturers instructions. Total of 200 mg of powdered sample was used. In the case of candied papaya the pro-tocol was modified as follows. The volume of lysis buffer increased to 700 µl and time of incubation was prolonged to 1 hour.

Assessment of DNA quality. DNAs were evalu-ated by agarose electrophoresis for its integrity and raugh estimation of quantity. For detailed


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Hasil (Bahasa Indonesia) 1:

BAHAN DAN METODEBahan. Pepaya sampel dikumpulkan di pasar. Buah kupas, daging terpisah dari batu, potong-potong dan irisan daging pepaya dikeringkan dalam oven di 37° C selama 12 h untuk mengurangi kadar air. Kedua jaringan disimpan pada –80 ° C sampai diproses lebih lanjut.Pepaya manisan disimpan di kamar tem-perature sampai diproses.Isolasi DNA. Dari setiap sampel dua inde-pendent isolations dilakukan. Sebelum isolasi DNA, sampel yang homogenised di dalam nitrogen cair.CTAB berbasis isolasi DNA. CTAB metode (setil trimethylammonium bromida) DNA isolota-tion dilakukan menurut standar ISO (EN ISO 21571:2002). Total 200 mg bubuk sampel digunakan untuk isolasi DNA. Diekstrak DNAs adalah eluted di 60 μL 0.1 × TE buffer.Isolasi DNA Wizard. DNAs diekstraksi dan dimurnikan menggunakan metode Wizard diubah. 200 mg bubuk sampel dicampur dengan 860 μL mentega buffer (5mM Tris-HCl, pH 8.0, 2mM EDTA, 150mM NaCl, SDS 1%). Campuran ini adalah vortexed. 100 μL dari 5M guanidin hidroklorida dan 40 μL proteinase K hydrochloride ditambahkan ke solusi, campuran adalah pada vortexed dan setelah itu diinkubasi 58° c selama 5 jam dengan occassionaly campuran-ing. Setelah inkubasi, campuran disentrifugasi min 10 jam 13 000 × g. 500 μL supernatant dipindahkan ke microtube baru dan 10 μL RNase ditambahkan ke sampel. Sampel lebih lanjut diinkubasi pada 65° C selama 10 menit. DNA solu-tion kemudian dimurnikan dengan Wizard DNA Clean-Up sistem (Promega, Madison, Amerika Serikat).Isolasi DNA GeneSpin Kit. DNA diambil menggunakan GeneSpin DNA ekstraksi Kit (Eurofins, Freiburg, Jerman) yang mengikuti petunjuk produsen. Total 200 mg bubuk sampel digunakan. Dalam kasus manisan pepaya pro-tocol diubah sebagai berikut. Volume lysis penyangga ditingkatkan 700 μL dan waktu inkubasi ini diperpanjang sampai 1 jam.Penilaian kualitas DNA. DNAs adalah evalu-mendatangkan oleh agarose Elektroforesis untuk estimasi dengan integritas dan raugh kuantitas. Untuk S2-76