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35
LAMPIRAN
Lampiran 1. Empat belas isolat cendawan yang diidentifikasi dari tanaman kakao
pada media PDA
a. Isolat Hasil Pemurnian
b. Isolat Hasil Perbanyakan
36
Lampiran 2. Komposisi Reagen PCR
a. PCR Mix (25 µl)
Komposisi : 2X Taq Plus PCR Master Mix 12,5µl
Primer ITS4 1µl
Primer ITS 5 1µl
ddH2O 5,5µl
Template DNA 5µl
b. Gel Agarose 2%
Komposisi : Serbuk Agarose 2 g
TBE Buffer 100 mL
Ethidium Bromida 8µl
Lampiran 3. Kit ekstraksi (TIANamp Genomic DNA kit ) yang digunakan dalam
ekstraksi DNA
37
Lampiran 4. Alur Kerja Ekstraksi DNA
Sampel (30 mg)
200 µl Buffer GA
20 µl Proteinase K
200 µl Buffer GB
200 µl Etanol
500 µl Buffer GD
600 µl Buffer PW
600 µl Buffer PW
200 µl Buffer TE
Sentrifius 2 menit
Gerus, sentrifius pada 11.200
G/1 menit
Vortex, inkubasi pada suhu
56⁰C/10 menit
Vortex, inkubasi pada suhu
70⁰C/10 menit
Pindahkan ke spin column,
sentrifius pada 13.400 G/30
detik
Sentrifius pada 13.400 G/30
detik
Sentrifius pada 13.400 G/30
detik
Pindahkan ke tube, sentrifius
pada 13.400 G/2 menit
Inkubasi 2 menit
38
Lampiran 5. Alur Kerja Amplifikasi DNA dengan PCR
Sampel DNA
Amplifikasi dengan
PCR
Produk PCR
Diamplifikasi sebanyak 35 siklus.
Setiap siklus terdiri dari:
Predenaturasi pada suhu 940C
selama 3 menit
Denaturasi pada suhu 940C
selama 30 detik
Annealing pada suhu 520C
selama 30 detik
Extending pada suhu 720C
selama 1 menit.
Post Extending pada suhu 720C
selama 5 menit.
20 µl campuran
reagen PCR
5 µl template DNA
Dibuatkan campuran reagen, yaitu
2X Taq Plus PCR Master Mix
12,5 µl, Primer ITS4 1 µl, Primer
ITS 5 1 µl, dan ddH2O 5,5 µl.
Dimasukkan ke dalam mikro
tube
Dimasukkan ke dalam mikro
tube yang berisi reagen PCR
39
Lampiran 6. Alur Kerja Elektroforesis Produk PCR
Produk PCR
Dibuatkan gel agarose 2%
dengan komposisi:
Serbuk Agarose 2 g
TBE Buffer 100 ml
Ethidium Bromida 8µl
5 µl produk PCR
Gel agarose 2%
Elektroforesis
Pita DNA
Dipipet dengan pipet mikron
dan dimasukkan ke dalam
masing-masing sumuran
Dielektroforesis dengan arus
listrik 70 V selama 30 menit.
Gel hasil elektroforesis
diletakkan di atas uv
transluminator dan difoto
untuk dokumentasi.
40
Lampiran 7. Foto Proses Kerja
Proses PCR
Proses Elektroforesis
41
Lampiran 8. Hasil Sekuensing Isolat LasioKB (Lasiodiplodia theobromae)
42
Lampiran 9. Hasil Sekuensing Isolat LasioKK1 (Lasiodiplodia brasiliensis)
43
Lampiran 10. Hasil Sekuensing Isolat LasioKK2 (Lasiodiplodia theobromae)
44
Lampiran 11. Hasil Sekuensing Isolat LasioKK3 (Tidak teridentifikasi)
45
Lampiran 12. Hasil Sekuensing Isolat LasioKL1 (Lasiodiplodia citricola)
46
Lampiran 13. Hasil Sekuensing Isolat LasioKL2 (Lasiodiplodia theobromae)
47
Lampiran 14. Hasil Sekuensing Isolat LasioKL3 (Lasiodiplodia citricola)
48
Lampiran 15. Hasil Sekuensing Isolat LasioKL4 (Lasiodiplodia citricola)
49
Lampiran 16. Hasil Sekuensing Isolat LasioKL5 (Lasiodiplodia theobromae)
50
Lampiran 17. Hasil Sekuensing Isolat LasioKL6 (Lasiodiplodia theobromae)
51
Lampiran 18. Hasil Sekuensing Isolat LasioKL7 (Lasiodiplodia theobromae)
52
Lampiran 19. Hasil Sekuensing Isolat LasioKL8 (Lasiodiplodia citricola)
53
Lampiran 20. Hasil Sekuensing Isolat LasioKL9 (Lasiodiplodia brasiliensis)
54
Lampiran 21. Hasil Sekuensing Isolat LasioKL10 (Lasiodiplodia theobromae)