lampiran 1. hasil amplifikasi dna menggunakan beberapa...

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Lampiran 1. Hasil Amplifikasi DNA Menggunakan Beberapa Jenis Primer OPA

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Lampiran 2. Protokol Isolasi DNA Genom Menggunakan Kit Wizard®

Genomic DNA Purification (Promega)

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Lampiran 3. Prosedur Pengukuran Kemurnian DNA Genom dengan

Spektrofotometrik

1. Menghubungkan steker spektrofotometrik dengan sumber tenaga, ubah

posisi saklar (tombol power) ke posisi ON.

2. Menunggu tampilan spektrofotometrik menjadi “smart start menu”,

masukkan blanko aquades (bagian bening menghadap ke depan).

3. Pilih “test DNA’ à enter à pilih “run test” à tekan tombol c à pilih

“measure blank” à tekan tombol a.

4. Keluarkan blanko aquades, dan masukan blanko sampel (bagian bening

menghadap ke depan).

5. Pilih “measure sample” à catat nilai yang muncul pada monitor à esc

(2x) sampai tampilan kembali ke “smart start menu”

6. Keluarkan blanko sampel bila pengukuran sudah selesai à matikan alat

dengan mengubah saklar (tombol power) ke posisi OFF.

Catatan:

· Blanko aquades terdiri dari 250 µl aquades

· Blanko sampel terdiri dari 5 µl sampel ditambah 245 µl aquades

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Lampiran 4. Hasil Amplifikasi DNA Sampel Ikan Cyprinid Uji

Menggunakan Primer OPA-5 dan OPA-13.

OPA-5

Keterangan:

M1 = Marker 1 kb SB = Ikan mas Subang

MJ = Ikan mas Majalaya GC = Grass carp

RD = Ikan mas Rajadanu GB = Giant barb

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Lampiran 5. Panjang Fragmen DNA (OPA-2)

Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp) 58.205 3.48 3000 62.935 3.40 2500 71.137 3.30 2000 78.185 3.24 1750 85.557 3.18 1500

Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp) 85.557 3.18 1500 97.142 3.10 1250

108.079 3.00 1000 117.033 2.94 875 126.171 2.88 750 139.35 2.80 625

152.761 2.70 500 171.458 2.57 375 190.432 2.40 250

Grafik Migrasi DNA (OPA-2)

y = -0.009x + 4.000R² = 0.993

3.103.203.303.403.50

0 20 40 60 80 100

Primer OPA-2

Jarak Fragment

Linear (Jarak Fragment)

y = -0.007x + 3.792R² = 0.997

0.001.002.003.004.00

0 50 100 150 200

Primer OPA-2

Jarak Fragment


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Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp) 69.818 3.30 2000 76.677 3.24 1750 83.644 3.18 1500 94.771 3.10 1250

105.969 3.00 1000 Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp)

69.818 3.30 2000 76.677 3.24 1750 85.557 3.18 1500 97.142 3.10 1250

107.668 2.98 950 116.335 2.93 850 124.934 2.86 725 137.303 2.80 625 149.822 2.70 500 167.895 2.57 375 186.053 2.40 250

Grafik Migrasi DNA (OPA-3)

y = -0.008x + 3.874R² = 0.998

2.80

3.00

3.20

3.40

0 20 40 60 80 100 120

Primer OPA-3

Jarak Fragment


y = -0.007x + 3.774R² = 0.993

0.00

1.00

2.00

3.00

4.00

0 50 100 150 200

Primer OPA-3

Jarak Fragment


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Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp) 66.211 3.40 2500 70.511 3.35 2250 74.414 3.30 2000 82.351 3.24 1750 90.024 3.18 1500

102.592 3.10 1250 114.895 3.00 1000

Jarak fragmen (mm) Log bp Antilog (INV-LOG) (bp) 85.557 3.18 1500 97.142 3.10 1250

114.895 2.98 950 124.552 2.90 800 134.209 2.88 750 148.695 2.80 625 162.784 2.70 500 184.15 2.57 375

205.118 2.40 250

Grafik Migrasi DNA OPA-13

y = -0.008x + 3.907R² = 0.992

2.803.003.203.403.60

0 50 100 150

OPA-13

Jarak Fragment


y = -0.006x + 3.700R² = 0.990

0.001.002.003.004.00

0 50 100 150 200 250

OPA-13

Jarak Fragment


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Lampiran 8. Interpretasi Pola Pita DNA (OPA-2)

Alel DNA Ukuran Basa Pita MJ RD SB GC GB

Alel 1 2514.94 0 0 0 1 0 Alel 2 1748.66 0 0 0 0 1

Alel 3 1180.56 0 0 0 1 0

Alel 4 1069.11 0 0 0 1 0

Alel 5 999.08 0 0 0 0 1 Alel 6 905.78 1 0 0 0 0 Alel 7 769.44 0 0 0 1 0

Alel 8 697.56 1 1 1 1 0

Alel 9 616.41 0 0 0 0 1

Alel 10 581.30 1 0 0 0 0 Alel 11 540.07 0 0 0 0 1 Alel 12 467.18 0 1 1 0 0

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Alel DNA

Ukuran Basa Pita MJ RD SB GC GB

Alel 1 1755.04 0 0 0 1 0

Alel 2 1639.29 0 0 0 0 1

Alel 3 1477.99 0 0 0 1 0

Alel 4 1337.46 1 0 0 0 0

Alel 5 1040.57 1 1 1 0 1

Alel 6 980.58 1 1 1 0 1

Alel 7 901.25 0 1 0 0 0

Alel 8 813.73 0 0 0 0 1

Alel 9 780.55 0 1 0 1 0

Alel 10 736.88 0 0 0 0 1

Alel 11 688.42 1 0 0 0 0

Alel 12 621.32 0 0 0 1 0

Alel 13 607.56 1 0 0 0 0

Alel 14 561.47 0 0 0 0 1

Alel 15 542.63 1 1 1 0 0

Alel 16 473.91 0 0 0 0 1

Alel 17 418.78 1 1 0 0 0

Alel 18 338.37 0 0 0 0 1

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Alel DNA

Ukuran Basa Pita MJ RD SB GC GB

Alel 1 1956.97 0 0 0 1 0

Alel 2 1872.99 0 1 1 0 0

Alel 3 1618.20 0 1 1 0 0

Alel 4 1416.94 0 0 0 0 1

Alel 5 1349.53 0 0 0 1 1

Alel 6 1177.39 0 0 0 0 1

Alel 7 1113.19 0 0 0 1 1

Alel 8 1073.26 0 0 0 0 1

Alel 9 1004.91 0 0 0 0 1

Alel 10 992.52 0 0 0 0 1

Alel 11 956.89 1 0 0 1 1

Alel 12 874.11 1 1 1 1 1

Alel 13 824.48 1 1 1 0 0

Alel 14 701.98 0 0 0 0 1

Alel 15 631.95 1 1 1 0 0

Alel 16 614.87 0 0 0 0 1

Alel 17 518.75 0 0 0 0 1

Alel 18 454.78 1 0 1 0 0

Alel 19 436.86 0 0 0 0 1

Alel 20 390.06 1 1 1 0 0

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Lampiran 11. Prosedur Analisis Data Menggunakan NTSYS-PC

1. Data pita hasil amplifikasi diubah menjadi data biner (1 dan 0), kemudian

dimasukkan ke dalam Microsoft Excel. Baris pertama (kolom A) diisi dengan

angka 1, baris pertama (kolom B) diisi dengan jumlah variabel yang diamati

(jumlah alel), baris pertama (kolom C) diisi dengan banyaknya jumlah sampel.

Data disimpan dalam format Microsoft Excel 1997 – 2003.

2. Membuka program NTEdit à pilih file à import à using OLE (pilih file

yang akan diimport) à save file as.

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3. Membuka program NTSYS-PC à pilih similarity (menganalisa kesamaan

genetik) à pilih program simqual (data matriks biner) à input:

Input file : Nama file data skoring berbentuk NTSys file By Rows : COL sebagai default, tidak perlu di klik Coefficient : DICE sebagai default koefisien kemiripan, dapat diganti

sesuai dengan kebutuhan Output file : Nama output hasil olahan Simqual Positive Code : 1 Negative Code : 0

4. Pilih menu clustering (mengolah data simqual menjadi dendogram/pohon

filogeni) à klik SAHN àààà input:

Name of input matrix : nama file hasil olahan Simqual Name of output matrix : nama file hasil olahan Clustering Method : UPGMA, default koefisien penghitungan, diganti dengan mengklik tanda panah In case of ties : WARN sebagai default diganti dengan FIND Maximum no. tied trees : default 25 Tie Tolerance : default 0 Show tree? : default YES Beta : default – 0,25 Listing File : default CON

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5. Output fenogram (pohon kekerabtan) dapat dilihat dengan mengklik kotak

fenogram (warna merah) pada menu SAHN (tanda à).

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Lampiran 12. Indeks Koefisien Kesamaan Genetik “Simple Matching”

Primer OPA-2 SimQual: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 6/16/2014 10:27:01 PM ---------------------------------------- Input parameters Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.NTS Compute by: cols Save results in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.1.NTS Coefficient: DICE Positive: 1.0000 Negative: 0.0000 type=1, size=12 by 5, nc=none (rectangular) Result will be a 5 by 5 matrix Results stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.1.NTS SAHN: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 6/16/2014 10:34:24 PM ---------------------------------------- Input parameters Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.1.NTS Save result tree in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.2.NTS Clustering method: UPGMA In case of ties: FIND Max. no. tied trees: 25 Comments: SIMQUAL: input=D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.NTS, coeff=DICE by Cols, += 1.00000, -= 0.00000 type=3, size=5 by 5, nc=none (similarity) Results will be stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-2\OPA-2.2.NTS Searching for all tied trees Solution tree number 1 Sorting tree nodes ... none needed. A single tree was found

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" SIMQUAL: input=D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\Corel RAPD\NTSYS\OPA-2.NTS, coeff=DICE " by Cols, += 1.00000, -= 0.00000 3 5L 5 0 MJ RD SB GC GB MJ 1.0000000 RD 0.4000000 1.0000000 SB 0.4000000 1.0000000 1.0000000 GC 0.2500000 0.2857143 0.2857143 1.0000000 GB 0.0000000 0.0000000 0.0000000 0.0000000 1.0000000 Primer OPA-3 SimQual: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 6/20/2014 9:41:01 PM ---------------------------------------- Input parameters Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.NTS Compute by: cols Save results in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.1.NTS Coefficient: DICE Positive: 1.0000 Negative: 0.0000 type=1, size=17 by 5, nc=none (rectangular) Result will be a 5 by 5 matrix Results stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.1.NTS SAHN: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 6/20/2014 9:41:13 PM ---------------------------------------- Input parameters Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.1.NTS Save result tree in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.2.NTS Clustering method: UPGMA In case of ties: FIND Max. no. tied trees: 25 Comments: SIMQUAL: input=D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.NTS, coeff=DICE by Cols, += 1.00000, -= 0.00000

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type=3, size=5 by 5, nc=none (similarity) Results will be stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.2.NTS Searching for all tied trees Solution tree number 1 Sorting tree nodes ... none needed. A single tree was found

" SIMQUAL: input=D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-3\OPA-3.NTS, coeff=DICE " by Cols, += 1.00000, -= 0.00000 3 5L 5 0 MJ RD SB GC GB MJ 1.0000000 RD 0.6666667 1.0000000 SB 0.6000000 0.8000000 1.0000000 GC 0.0000000 0.2000000 0.0000000 1.0000000 GB 0.2857143 0.2857143 0.3333333 0.0000000 1.0000000

Primer OPA-13

SimQual: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 7/3/2014 12:26:25 AM ---------------------------------------- Input parameters Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.NTS Compute by: cols Save results in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.1.NTS Coefficient: DICE Positive: 1.0000 Negative: 0.0000 type=1, size=20 by 5, nc=none (rectangular) Result will be a 5 by 5 matrix Results stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.1.NTS SAHN: NTSYSpc 2.02, (C) 1986-1998, Applied Biostatistics Inc. Date & time: 7/3/2014 12:27:01 AM ---------------------------------------- Input parameters

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Read input from file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.1.NTS Save result tree in output file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.2.NTS Clustering method: UPGMA In case of ties: FIND Max. no. tied trees: 25 Comments: SIMQUAL: input=D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.NTS, coeff=DICE by Cols, += 1.00000, -= 0.00000 type=3, size=5 by 5, nc=none (similarity) Results will be stored in file: D:\Kuli\000 - TA Skripsi\00 - RAPD Carp\Doc\01 - Corel RAPD\NTSYS\OPA-13\OPA-13.2.NTS Searching for all tied trees Solution tree number 1 Sorting tree nodes ... none needed. A single tree was found

lampiran 1. hasil amplifikasi dna menggunakan beberapa...

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