Sampel DNA kelapa sawit (normal dan abnormal) Isolasi DNA sampel. Proses PCR-RAPD. Sequencing. Analisis hasil sequencing

Transkripsi

1 LAMPIRAN

2 16 Lampiran 1 Alur penelitian Sampel DNA kelapa sawit (normal dan abnormal) Isolasi DNA sampel Proses PCR-RAPD Isolasi fragmen hasil PCR-RAPD Sequencing Analisis hasil sequencing

3 17 Lampiran 2 Pembuatan larutan sediaan Pembuatan bufer ekstraksi DNA. (50 ml) Bufer ekstraksi DNA dibuat dengan mencampurkan : CTAB 10% 10 ml EDTA 0.5 M ph ml tris-hcl 1M ph ml NaCl 5 M 12.6 ml dh 2 O steril 20.4 ml Pembuatan Bufer TE (100 ml) Bufer TE dibuat dengan melarutkan : Tris-HCl 1 M ph ml EDTA 0.5 M ph ml dh 2 O steril 98.8 ml Pembuatan Bufer TBE 5 (500 ml) Bufer TBE 5 dibuat dengan komposisi : Tris-Base 27gram Asam borat gram EDTA 0.5 M ph ml dan ditepatkan dengan ddh 2 O hingga 500 ml. Saat pemakaian dalam pembuatan gel agarosa, bufer TBE 5 ini diencerkan menjadi 0.5.

4 18 Lampiran 3 Proses isolasi DNA daun, bunga, dan buah kelapa sawit Sterilisasi alat dan bahan dengan otoklaf (121 C, 1 atm) Masing-masing sampel ditimbang sebanyak 1 gram Sampel digerus dengan mortar Ditambah 0.1 g PVPP dan N 2 cair sampai halus Tabung sentrifus berisi 5 ml bufer ekstraksi dan ß-merkaptoetanol yang sudah dipanaskan (65 C) Dipanaskan pada 65 C selama 30 menit (tiap 5 menit dikocok) Larutan didinginkan pada suhu kamar Diekstraksi dengan larutan CI sebanyak 1 volume Homogenasi dengan vorteks Disentrifugasi pada ±13500 g, 10 menit, 4 C Supernatan dipindahkan ke tabung baru Diulang 2 Diekstraksi dengan larutan CI sebanyak 1 volume Ditambah isopropanol dingin 1 volume Disimpan dalam lemari es 4 C, 30 menit Disentrifugasi pada ±13500 g, 10 menit, 4 C Pelet DNA dikeringkan Ditambah 1 ml bufer TE Kocok hingga pelet DNA semua larut Ditambah Na-asetat 1/10 volume dan etanol absolut 2.5 ml Dibekukan selama 30 menit atau semalam

5 19 Lanjutan Lampiran 3 Disentrifugasi pada ±16000 g, 10 menit, 4 C Pelet DNA dicuci dengan etanol 70% Pelet dikeringkan dengan sentrifugasi pada ±16000 g, 5 menit, 4 C Pelet dilarutkan dalam MW Dipindahkan dalam tabung ependorf (DNA stok)

6 20 Lampiran 4 Perhitungan pengenceran DNA dengan konsentrasi 100 ng/µl V2 M 2 V 1 M 1 = V 2 M 2 V 1 = M Keterangan: V 1 = volume larutan DNA yang diambil untuk diencerkan M 1 = konsentrasi larutan DNA sebelum diencerkan V 2 = volume akhir pengenceran (50 μl) M 2 = konsentrasi larutan DNA yang diinginkan (100 ng/μl) 1 Volume DNA yang diencerkan (µl) V 1 (DNA Daun N) = = V 1 (DNA Daun Ab) = = V 1 (DNA Bunga N) = = V 1 (DNA Bunga Ab) = = V 1 (DNA Buah N) = = V 1 (DNA Buah Ab) = = Penambahan MW (µl)

7 21 Lampiran 5 Tabel urutan basa primer OPA dan OPB No Kode 5 to 3 1 OPA 6 GGT CCC TGAC 2 OPA 7 GAA ACG GGTG 3 OPA 8 GTG ACG TAGG 4 OPA 9 GGG TAA CGCC 5 OPA 10 GTG ATC GCAG 6 OPA 14 TCT GTG CTGG 7 OPA 15 TTC CGA ACCC 8 OPA 17 GAC CGC TTGT 9 OPA 18 AGG TGA CCGT 10 OPA 19 CAA ACG TCGG 11 OPA 20 GTT GCG ATCC 12 OPB 1 GTT TCG CTCC 13 OPB 2 TGA TCC CTGG 14 OPB 3 CAT CCC CCTG 15 OPB 7 GGT GAC GCAG 16 OPB 8 GTC ACA ACGG 17 OPB 9 TGG GGG ACTC 18 OPB 10 CTG CTG GGAC 19 OPB 17 AGG GAA CGAG Lampiran 6 Gambar pola pita hasil amplifikasi DNA

8 M Keterangan gambar: (1) DN (2) Dab (3) BN (4) BAb (5) FN (6) FAb (M) Marker 1 kb + OPA 6 OPA 7 a. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPA 6 dan OPA M b. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPA 14 dan OPA M M c. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer Lanjutan OPA 10. Lampiran 6 d. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPA 17.

9 M M Keterangan gambar: (1) DN (2) Dab (3) BN (4) BAb (5) FN (6) FAb (M) Marker 1 kb + e. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPB 1. f. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPB M M g. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPB 7. h. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPB M OPB 8 OPB 17 i. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer OPB 8 dan OPB 17. Lampiran 7 Hasil analisis BLAST dari urutan basa bunga abnormal dengan primer OPB 9

10 24 Deskripsi Max Total E Max score score value ident Mus musculus strain 129S6/SvEvTac clone rp21-580p % map 10, Mus musculus strain C57BL/6J clone rp23-358m4, % Mus musculus strain 129S6/SvEvTac chromosome 10 clone % rp21-411d9, Canis lupus familiaris clone RP81-386E20, complete % Mus musculus chromosome 6, clone RP23-402P24, % Mus musculus chromosome 5, clone RP24-67M11, % Mus musculus chromosome 5, clone RP23-445H19, % Drosophila melanogaster chromosome 3R, complete % Mus musculus BAC clone RP23-119P9 from chromosome % 13, Drosophila melanogaster clone BACR03J17, complete % Drosophila melanogaster clone BACR27G04, complete % Mus musculus BAC clone RP24-422I4 from chromosome % 13, Mustela putorius furo clone CH F7, complete % Felis catus clone RP86-181L4, % Mus musculus BAC clone RP23-473K23 from chromosome % 13, Canis familiaris, clone XX-397C16, % Canis Familiaris chromosome 12, clone XX-93M5, % Canis Familiaris chromosome 3, clone XX-500D6, complete % Canis Familiaris chromosome 14, clone XX-483F1, % Canis Familiaris chromosome 6, clone XX-463G11, % Canis Familiaris chromosome 3, clone XX-393O18, % Canis lupus familiaris clone RP81-196D8, complete % Canis Familiaris, clone XX-10C7, % Mus musculus chromosome 3, clone RP23-296I12, % Human DNA from clone RP11-92B10 on % chromosome X Contains the 5' end of a variant of the CUL4B gene for cullin 4B and the 5' end of a variant of the gene for MCT-1 protein (MCT-1), Mus musculus chromosome 11, clone RP23-179C3, % Lanjutan Lampiran 7 Deskripsi Max score Total score E value Max ident

11 25 Mus musculus BAC clone RP23-25I3 from chromosome 7, % Mouse DNA from clone RP23-285D3 on % chromosome 11 Contains part of a novel gene, a heat shock protein (Hsp) pseudogene and a CpG island, complete Mouse DNA from clone RP24-246F8 on % chromosome 13, Mouse DNA from clone RP24-120H1 on % chromosome 16, Mouse DNA from clone RP23-406N5 on % chromosome 4 Contains a ribosomal protein S19 (Rps19) pseudogene, a novel gene, the gene for a DNA segment Chr 4 Wayne State University 114 expressed (D4Wsu114e), the Mfn2 gene for mitofusin 2, a ribosomal protein L35a (Rpl35a) pseudogene, the Plod1 gene for procollagen-lysine 2-oxoglutarate 5-dioxygenase 1, a novel gene ( O18Rik) and two CpG islands, Dictyostelium discoideum AX4 chromosome % DDB , whole genome shotgun Canis familiaris chromosome 33, clone XX-221P23, % Monosiga brevicollis MX1 predicted protein % MONBRDRAFT_7962 mrna, complete cds Mouse DNA from clone RP23-102L17 on % chromosome 16, Canis Familiaris chromosome 26, clone XX-195P12, % Canis Familiaris chromosome 25, clone XX-122B22, % Vitis vinifera, whole genome shotgun, contig % VV78X , clone ENTAV 115 Canis Familiaris chromosome 26, clone XX-497F13, % Canis Familiaris chromosome 37, clone XX-341P11, % Canis Familiaris chromosome 20, clone XX-263M4, % Canis Familiaris chromosome 26, clone XX-320A4, % Canis Familiaris chromosome 33, clone XX-84F14, % Mus musculus chromosome 15, clone RP23-85P16, % Canis Familiaris, clone XX-25G10, % Felis catus clone RP86-439N5, % Mus musculus chromosome 15, clone RP23-338F23, % Mus musculus BAC clone RP24-241I3 from 15, complete % Zebrafish DNA from clone DKEY-83M22 in linkage group 9, % Lanjutan Lampiran 7 Max Total E Max Deskripsi score score value ident Arabidopsis thaliana DNA chromosome 3, BAC clone %

12 26 T28A8 Mouse DNA from clone DN-316O2 on % chromosome 3 Contains two ribosomal protein, large, P0 (Rplp0) pseudogene, Mus musculus genomic DNA, chromosome 16q % clone:rp21-567f3, s Dictyostelium discoideum AX4 chromosome % DDB , whole genome shotgun Dictyostelium discoideum AX4 chromosome % DDB , whole genome shotgun Canis familiaris chromosome 26, clone XX-397I18, % Populus trichocarpa clone POP037-N11, % Canis familiaris chromosome 14, clone XX-54L5, complete % Canis familiaris chromosome 2, clone XX-83E16, complete % Canis familiaris chromosome 26, clone XX-93E11, % Zebrafish DNA from clone CH211-51E8 in % linkage group 21, Lodderomyces elongisporus NRRL YB-4239 chromo % domain protein 1 (LELG_01549) partial mrna Canis familiaris chromosome 31, clone XX-361C3, % Tetrahymena thermophila SB210 hypothetical protein % (TTHERM_ ) partial mrna Canis Familiaris chromosome 10, clone XX-480A12, % Canis Familiaris, clone XX-284L8, % Canis Familiaris chromosome 11, clone XX-311M9, % Vitis vinifera contig VV78X , whole genome % shotgun Canis Familiaris chromosome 10, clone XX-181J11, % Canis Familiaris chromosome 14, clone XX-498K4, % Canis Familiaris chromosome 16, clone XX-195I11, % Canis Familiaris chromosome 8, clone XX-494F18, % Canis Familiaris chromosome 2, clone XX-361N13, % Canis Familiaris chromosome X, clone XX-242J14, % Canis Familiaris chromosome 6, clone XX-346D24, % Zebrafish DNA from clone RP71-84K24 in % linkage group 3, Canis Familiaris chromosome 8, clone XX-368A9, % Lanjutan Lampiran 7 Deskripsi Canis Familiaris chromosome 6, clone XX-163J21, Max Total E Max score score value ident %

13 27 Canis Familiaris chromosome 32, clone XX-3D7, complete % Canis Familiaris chromosome 32, clone XX-98B12, % Xenopus (Silurana) tropicalis chromosome CH216-62P2, % Dictyostelium discoideum AX4 component of % complex (rad9) partial mrna Canis lupus familiaris agouti signaling protein, nonagouti % homolog (mouse) (ASIP), mrna >gb AY Canis familiaris agouti protein mrna, complete cds Mus musculus BAC clone RP24-178A16 from chromosome % 16, Mus musculus BAC clone RP23-227O11 from chromosome % 12, Canis lupus familiaris clone RP81-55A4, complete % Homo sapiens chromosome 5 clone CTD-2170G1, % Homo sapiens BAC clone RP11-436F23 from 4, complete % Felis sp. NG192 DNA, INT1095 locus % Homo sapiens chromosome 5 clone RP11-164A5, complete % Homo sapiens chromosome 5 clone RP11-54A24, complete % Mouse DNA from clone RP23-137B16 on % chromosome 11 Contains a novel gene, the 5' end of the Meis1 gene for myeloid ecotropic viral integration site 1 and two Cpg islands, Dictyostelium discoideum chromosome 2 map % strain AX4, Mus musculus strain C57BL/6J chromosome 2 clone rp % 60i12, Mouse DNA from clone RP23-259G10 on % chromosome 9, Mouse DNA from clone RP23-300E5 on % chromosome 2 Contains the Lrp2 gene for low density lipoprotein receptor-related protein 2 and a CpG island, Mus musculus BAC clone RP23-387C9 from 12, complete % Mouse DNA from clone RP23-393E22 on % chromosome 16, Mus musculus BAC clone RP23-299A14 from chromosome 6, %