Lampiran 1 Alur penelitian
Sampel DNA kelapa sawit (normal dan abnormal)
Proses PCR-RAPD
Isolasi fragmen hasil PCR-RAPD
Sequencing
Analisis hasil sequencing
Isolasi DNA sampel
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 8.0
2 ml
tris-HCl 1M pH 8.0
5 ml
NaCl 5 M
12.6 ml
dH
2O steril
20.4 ml
Pembuatan Bufer TE (100 ml)
Bufer TE dibuat dengan melarutkan :
Tris-HCl 1 M pH 8.0
1 ml
EDTA 0.5 M pH 8.0
0.2 ml
dH
2O steril
98.8 ml
Pembuatan Bufer TBE 5× (500 ml)
Bufer TBE 5× dibuat dengan komposisi :
Tris-Base
27gram
Asam borat
13.75 gram
EDTA 0.5 M pH 8.0
10 ml
dan ditepatkan dengan ddH
2O hingga 500 ml.
Saat pemakaian dalam pembuatan gel agarosa, bufer TBE 5× ini diencerkan
menjadi 0.5×.
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
2cair 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
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
Lampiran 4 Perhitungan pengenceran DNA dengan konsentrasi 100 ng/µl
V
1×M
1= V
2×M
2→ V
1=
1 2 2M
M
×
V
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)
Volume DNA yang diencerkan (µl)
Penambahan MW (µl)
V
1(DNA Daun N) =
6
.
09
820
100
50
=
×
43.91
V
1(DNA Daun Ab) =
5
.
78
865
100
50
=
×
44.22
V
1(DNA Bunga N) =
4
.
28
1180
100
50
=
×
45.72
V
1(DNA Bunga Ab) =
3
.
86
1295
100
50
×
=
46.14
V
1(DNA Buah N) =
6
.
25
800
100
50
×
=
43.75
V
1(DNA Buah Ab) =
5
.
58
895
100
50
×
=
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
1 2 3 4 5 6 1 2 3 4 5 6 M
OPA 6 OPA 7
1 2 3 4 5 6 M 1 2 3 4 5 6
1 2 3 4 M 5 6
1 2 3 4 5 6 M
Lanjutan Lampiran 6
Keterangan gambar: (1) DN (2) Dab (3) BN (4) BAb (5) FN (6) FAb (M) Marker 1 kb +a. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer
OPA 6 dan OPA 7.
c. Sampel DNA daun, bunga, dan buah
normal dan abnormal dengan primer
OPA 10.
d. Sampel DNA daun, bunga, dan buah
normal dan abnormal dengan primer
OPA 17.
b. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer
OPA 14 dan OPA 15.
1 2 3 4 5 6 M 1 2 3 4 5 6 M
1 2 3 4 5 6 M M 1 2 3 4 5 6
1 2 3 4 5 6 1 2 3 4 5 6 M
OPB 8
OPB 17
Lampiran 7 Hasil analisis BLAST dari urutan basa bunga abnormal dengan primer
OPB 9
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 3.
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 10.
i. Sampel DNA daun, bunga, dan buah normal dan abnormal dengan primer
OPB 8 dan OPB 17.
Deskripsi Max score Total score E value Max ident
Mus musculus strain 129S6/SvEvTac clone rp21-580p20 map 10, complete sequence
51.8 51.8 0.003 80%
Mus musculus strain C57BL/6J clone rp23-358m4, complete sequence
51.8 51.8 0.003 80%
Mus musculus strain 129S6/SvEvTac chromosome 10 clone rp21-411d9, complete sequence
51.8 51.8 0.003 80%
Canis lupus familiaris clone RP81-386E20, complete sequence
50.0 50.0 0.011 78%
Mus musculus chromosome 6, clone RP23-402P24, complete sequence
50.0 50.0 0.011 76%
Mus musculus chromosome 5, clone RP24-67M11, complete sequence
50.0 50.0 0.011 79%
Mus musculus chromosome 5, clone RP23-445H19, complete sequence
50.0 50.0 0.011 79%
Drosophila melanogaster chromosome 3R, complete sequence
48.2 48.2 0.039 79%
Mus musculus BAC clone RP23-119P9 from chromosome 13, complete sequence
48.2 48.2 0.039 80%
Drosophila melanogaster clone BACR03J17, complete sequence
48.2 48.2 0.039 79%
Drosophila melanogaster clone BACR27G04, complete sequence
48.2 48.2 0.039 79%
Mus musculus BAC clone RP24-422I4 from chromosome 13, complete sequence
48.2 48.2 0.039 80%
Mustela putorius furo clone CH237-388F7, complete sequence
46.4 46.4 0.14 76%
Felis catus clone RP86-181L4, complete sequence 46.4 90.9 0.14 76%
Mus musculus BAC clone RP23-473K23 from chromosome 13, complete sequence
46.4 46.4 0.14 80%
Canis familiaris, clone XX-397C16, complete sequence 44.6 44.6 0.47 77% Canis Familiaris chromosome 12, clone XX-93M5,
complete sequence
44.6 44.6 0.47 79%
Canis Familiaris chromosome 3, clone XX-500D6, complete sequence
44.6 44.6 0.47 77%
Canis Familiaris chromosome 14, clone XX-483F1, complete sequence
44.6 44.6 0.47 76%
Canis Familiaris chromosome 6, clone XX-463G11, complete sequence
44.6 44.6 0.47 76%
Canis Familiaris chromosome 3, clone XX-393O18, complete sequence
44.6 44.6 0.47 77%
Canis lupus familiaris clone RP81-196D8, complete sequence
44.6 44.6 0.47 76%
Canis Familiaris, clone XX-10C7, complete sequence 44.6 44.6 0.47 76% Mus musculus chromosome 3, clone RP23-296I12,
complete sequence
44.6 44.6 0.47 78%
Human DNA sequence 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), complete sequence
44.6 44.6 0.47 85%
Mus musculus chromosome 11, clone RP23-179C3, complete sequence 44.6 44.6 0.47 75%
Lanjutan Lampiran 7
Deskripsi Max score Total score E value Max identMus musculus BAC clone RP23-25I3 from chromosome 7, complete sequence
44.6 44.6 0.47 79%
Mouse DNA sequence from clone RP23-285D3 on
chromosome 11 Contains part of a novel gene, a heat shock protein (Hsp) pseudogene and a CpG island, complete sequence
44.6 44.6 0.47 75%
Mouse DNA sequence from clone RP24-246F8 on chromosome 13, complete sequence
44.6 44.6 0.47 79%
Mouse DNA sequence from clone RP24-120H1 on chromosome 16, complete sequence
44.6 44.6 0.47 86%
Mouse DNA sequence 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
(2510039O18Rik) and two CpG islands, complete sequence
44.6 44.6 0.47 76%
Dictyostelium discoideum AX4 chromosome 3 DDB0232519.02, whole genome shotgun sequence
42.8 42.8 1.6 76%
Canis familiaris chromosome 33, clone XX-221P23, complete sequence
42.8 42.8 1.6 75%
Monosiga brevicollis MX1 predicted protein MONBRDRAFT_7962 mRNA, complete cds
42.8 42.8 1.6 90%
Mouse DNA sequence from clone RP23-102L17 on chromosome 16, complete sequence
42.8 42.8 1.6 82%
Canis Familiaris chromosome 26, clone XX-195P12, complete sequence
42.8 42.8 1.6 81%
Canis Familiaris chromosome 25, clone XX-122B22, complete sequence
42.8 42.8 1.6 77%
Vitis vinifera, whole genome shotgun sequence, contig VV78X193509.12, clone ENTAV 115
42.8 42.8 1.6 78%
Canis Familiaris chromosome 26, clone XX-497F13, complete sequence
42.8 42.8 1.6 83%
Canis Familiaris chromosome 37, clone XX-341P11, complete sequence
42.8 42.8 1.6 75%
Canis Familiaris chromosome 20, clone XX-263M4, complete sequence
42.8 42.8 1.6 80%
Canis Familiaris chromosome 26, clone XX-320A4, complete sequence
42.8 42.8 1.6 80%
Canis Familiaris chromosome 33, clone XX-84F14, complete sequence
42.8 42.8 1.6 75%
Mus musculus chromosome 15, clone RP23-85P16, complete sequence
42.8 42.8 1.6 85%
Canis Familiaris, clone XX-25G10, complete sequence 42.8 42.8 1.6 76%
Felis catus clone RP86-439N5, complete sequence 42.8 42.8 1.6 78%
Mus musculus chromosome 15, clone RP23-338F23, complete sequence
42.8 42.8 1.6 85%
Mus musculus BAC clone RP24-241I3 from 15, complete sequence
42.8 42.8 1.6 77%
Zebrafish DNA sequence from clone DKEY-83M22 in linkage group 9, complete sequence
42.8 42.8 1.6 87%
Lanjutan Lampiran 7
Deskripsi Max score Total score E value Max identT28A8
Mouse DNA sequence from clone DN-316O2 on chromosome 3 Contains two ribosomal protein, large, P0 (Rplp0) pseudogene, complete sequence
42.8 42.8 1.6 72%
Mus musculus genomic DNA, chromosome 16q clone:RP21-567F3, complete sequences
42.8 42.8 1.6 82%
Dictyostelium discoideum AX4 chromosome 2 DDB0232469.02, whole genome shotgun sequence
41.0 41.0 5.7 93%
Dictyostelium discoideum AX4 chromosome 1 DDB0232442.02, whole genome shotgun sequence
41.0 41.0 5.7 74%
Canis familiaris chromosome 26, clone XX-397I18, complete sequence
41.0 41.0 5.7 73%
Populus trichocarpa clone POP037-N11, complete sequence 41.0 41.0 5.7 80% Canis familiaris chromosome 14, clone XX-54L5, complete
sequence
41.0 41.0 5.7 74%
Canis familiaris chromosome 2, clone XX-83E16, complete sequence
41.0 41.0 5.7 76%
Canis familiaris chromosome 26, clone XX-93E11, complete sequence
41.0 41.0 5.7 73%
Zebrafish DNA sequence from clone CH211-51E8 in linkage group 21, complete sequence
41.0 41.0 5.7 82%
Lodderomyces elongisporus NRRL YB-4239 chromo domain protein 1 (LELG_01549) partial mRNA
41.0 41.0 5.7 81%
Canis familiaris chromosome 31, clone XX-361C3, complete sequence
41.0 41.0 5.7 72%
Tetrahymena thermophila SB210 hypothetical protein (TTHERM_00188900) partial mRNA
41.0 41.0 5.7 100%
Canis Familiaris chromosome 10, clone XX-480A12, complete sequence
41.0 41.0 5.7 75%
Canis Familiaris, clone XX-284L8, complete sequence 41.0 41.0 5.7 80% Canis Familiaris chromosome 11, clone XX-311M9,
complete sequence
41.0 41.0 5.7 80%
Vitis vinifera contig VV78X194357.5, whole genome shotgun sequence
41.0 41.0 5.7 87%
Canis Familiaris chromosome 10, clone XX-181J11, complete sequence
41.0 41.0 5.7 75%
Canis Familiaris chromosome 14, clone XX-498K4, complete sequence
41.0 41.0 5.7 74%
Canis Familiaris chromosome 16, clone XX-195I11, complete sequence
41.0 41.0 5.7 75%
Canis Familiaris chromosome 8, clone XX-494F18, complete sequence
41.0 41.0 5.7 83%
Canis Familiaris chromosome 2, clone XX-361N13, complete sequence
41.0 41.0 5.7 75%
Canis Familiaris chromosome X, clone XX-242J14, complete sequence
41.0 41.0 5.7 75%
Canis Familiaris chromosome 6, clone XX-346D24, complete sequence
41.0 41.0 5.7 74%
Zebrafish DNA sequence from clone RP71-84K24 in linkage group 3, complete sequence
41.0 41.0 5.7 76%
Canis Familiaris chromosome 8, clone XX-368A9, complete sequence 41.0 41.0 5.7 82%
Lanjutan Lampiran 7
Deskripsi Max score Total score E value Max identCanis Familiaris chromosome 6, clone XX-163J21, complete sequence
Canis Familiaris chromosome 32, clone XX-3D7, complete sequence
41.0 41.0 5.7 78%
Canis Familiaris chromosome 32, clone XX-98B12, complete sequence
41.0 41.0 5.7 78%
Xenopus (Silurana) tropicalis chromosome CH216-62P2, complete sequence
41.0 41.0 5.7 92%
Dictyostelium discoideum AX4 component of 9-1-1 complex (rad9) partial mRNA
41.0 41.0 5.7 93%
Canis lupus familiaris agouti signaling protein, nonagouti homolog (mouse) (ASIP), mRNA >gb|AY714374.1| Canis familiaris agouti protein mRNA, complete cds
41.0 41.0 5.7 76%
Mus musculus BAC clone RP24-178A16 from chromosome 16, complete sequence
41.0 41.0 5.7 78%
Mus musculus BAC clone RP23-227O11 from chromosome 12, complete sequence
41.0 41.0 5.7 79%
Canis lupus familiaris clone RP81-55A4, complete sequence
41.0 41.0 5.7 74%
Homo sapiens chromosome 5 clone CTD-2170G1, complete sequence
41.0 41.0 5.7 92%
Homo sapiens BAC clone RP11-436F23 from 4, complete sequence
41.0 41.0 5.7 75%
Felis sp. NG192 DNA, INT1095 locus sequence 41.0 41.0 5.7 82%
Homo sapiens chromosome 5 clone RP11-164A5, complete sequence
41.0 41.0 5.7 92%
Homo sapiens chromosome 5 clone RP11-54A24, complete sequence
41.0 41.0 5.7 92%
Mouse DNA sequence 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, complete sequence
41.0 41.0 5.7 76%
Dictyostelium discoideum chromosome 2 map 1685067-2090751 strain AX4, complete sequence
41.0 41.0 5.7 93%
Mus musculus strain C57BL/6J chromosome 2 clone rp23-60i12, complete sequence
41.0 41.0 5.7 78%
Mouse DNA sequence from clone RP23-259G10 on chromosome 9, complete sequence
41.0 41.0 5.7 79%
Mouse DNA sequence from clone RP23-300E5 on chromosome 2 Contains the Lrp2 gene for low density lipoprotein receptor-related protein 2 and a CpG island, complete sequence
41.0 41.0 5.7 78%
Mus musculus BAC clone RP23-387C9 from 12, complete sequence
41.0 41.0 5.7 92%
Mouse DNA sequence from clone RP23-393E22 on chromosome 16, complete sequence
41.0 41.0 5.7 78%
Mus musculus BAC clone RP23-299A14 from chromosome 6, complete sequence
