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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)