lampiran 1. pembuatan baban ml. - unri.ac.id
TRANSCRIPT
Lampiran 1. Pembuatan Baban
1. Bufer tris-base 0,5 MpH 8 (Mr: 121,144)
Sebanyak 1,5143 gram tris-base dilarutkan dengan 20 mL aquades, dan diatur pH
menjadi 8 dengan penambahan HCl atau NaOH. Kemudian diencerkan hingga 25
mL.
2. Larutan EDTA 0,5 M (Mr: 292,504)
Sebanyak 3,6563 gram Na-EDTA dilarutkan dengan 25 mL aquad^
3. Bufer TAE 50 x kuat sebagai stok
Sebanyak 24,2 gram tris-base + 5,71 mL C H 3 C O O H glasial + 10 mL EDTA 0,5 M
pH 8 dicampurkan dengan 80 mL aquades, kemudian di^cerkan hingga 100 mL.
4. Loading buter
Dicampurkan 500 jiL gliserol 10 % + 333 jiL TAE 10 x kuat + 100 |iL bromophenol
blue 0,1 % (0,001 gram dilarutkan dengan 1 mL aquades).
5. Gel agarosa 0,8 % dalam TAE 1 x kuat
Sebanyak 0,24 gram agarosa dilarutkan dengan 30 mL TAE 1 x kuaL
6. Gel agarosa 1,2 % dalam TAE 1 x kuat
Sebanyak 0,36 gram agarosa dilarutkan dengan 30 mL TAE 1 x kuat
7. BuferO,lMNa-fosfatpH7
Larutan A : 1 M lanitan Na2HP04 (17,79 gram dalam 100 mL, Mr: 177,9)
Larutan B : 1 M larutan NaH2P04 (13,79 gram dalam 100 mL, Mr: 137,9)
Untuk membuat larutan bufer pH 7 sebanyak 100 mL, maka perbandingan larutan
yang dicampurkan sesuai tabel berikut:
No. pH Na2HP04lM(mL) NaH2P04lM(mL)
1. 6,0 1,20 8,80
2. 6,2 1,78 8,22
3. 6,4 2,55 7,45
4. 6,6 3,52 6,48
5. 6,8 4,63 5,37
6. 7,0 5,77 4,23
7. 7,2 6,84 3,16
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Lampiran 2. Kurva hubungan jarak migrasi standar DNA terhadap log pb
1. Hasil elektroforesis gambar 9 (penentuan berat molekul DNA hasil isolasi)
2. Hasil elektroforesis gambar 11 (Penentuan berat molekul DNA hasil PCR)
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Lampiran 3. Data penentuan titik leleh (Tm)
No. Temperatur ( ) Absorbans (A 260) Absorbans Relatif I. 30 0,163 1,000 2. 32 0,208 1,276 3. 34 0,235 1,442 4. 36 0,610 3,742 5. 38 0,632 3,877 6. 40 0,656 4,025 7. 42 0,679 4,166 8. 44 0,681 4,178 9. 46 0,689 4,227 10. 48 0,700 4,294 11. 50 0,705 4,325 12. 52 0,701 4,301 13. 54 0,700 4,294 14. 56 0,720 4,417 15. 58 0,738 4,528 16. 60 0,762 4,675 17. 62 0,785 4,816 18. 64 0,879 5,393 19. 66 0,935 5,736 20. 68 0,972 5,%3 21. 70 0,945 5,798 22. 72 0,965 5,920 23. 74 0,930 5,706 24. 76 0,971 5,957 25. 78 1,061 6,509 26. 80 1,095 6,718 27. 82 1,130 6,933 28. 84 1,143 7,012 29. 86 1,197 7,344 30. 88 0,906 5,558 31. 90 0,958 5,877
Keterangan: Absorbans relatif = A260 (pada suhu T leleh)
A 2 6 0 (p^a suhu kamar)
Contoh perhitungan (30 "C) : Absorbans relatif
Absorbans relatif
0,163
= 1,000
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Lampiran 4. Hasil printout pembacaan sistem sekuensing sebelum diperbaiki
1. Hasil sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer 1TS5
5' - NNNNNNNNNNNNNNNNCGNNNGGNNCATTACCGAGTTTACACTCCNNNNCCAN TGTGNNN^4NNACCAAACTGTTNCCTCGGCGGGGTCACGCCCCGGGTGCGTCGCAN CCCCGGAACCAGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTC GCGGACGTATTTCTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACA ACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGT GAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTA TTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGA T C G G C G T T G G G G A T C G G G A C C C C T C A C A C G G G T G N C G G C C C C N A A A T A C A G N G G C GGTCTCGCCGCANCCCTCTCCTGCGCANTANTTTGCACAACTCNNACNGGGAGCGC GGCGCGTCCACNTCCGTAAAACANCCAANTTTCTGAANTGTNNACCTNCNNATCANG NAAGANNNNNCGCTGAACNTNATCCNNNTCNATNNNNNGNANGANNNNNN - 3'
2. Hasil sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer ITS4
5' - N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N G N N N N N N N N N N NNNNNNNNNNCNTNNNNNCNGNCCNNNNCNCNTTTCNANNNTGGGTGTTTNNNNNN N N N N N N N N N N N N N N N G N G C G A G N N C C N C N N G C A A C N N N N N N N N N N N A G G N N N N N N N N N N N N N N N N N N N N N N T N N N N N N N N C N N N N N N N N N N N N N N G G N N N N N N N C N N N N N N N N N C N C N N N N N N N N N N N N N N N N N N A - 3'
3. Hasil sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer ITS3
5' - NNNNNNNNNNNNGGNGNNTTGNNNATTCNGTGATCrfTCGAATCTTTGACGCAC ATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTC G A A C C C C T C C G G G G G A T C G G C G T T G G G G A T C G G G A C C C C T C A C A C G G G T G C C G G CCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGTTTGCACAA CTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGAAATG TTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCNNNNNNNTNAA6CGGA GNNNNNNNCCNNNNNNNNNNTNNNNNAANNTTTTNNNNNNNTTTGAANNNNNNNNN N N A A N N N N N A N A A A N N A N N N N N N N N N N N N N N N N N N N N N N N N N N N N N T N N N N N G G N N N N N N N N N N G G N N N N N C C N G N N N N C N N N N N N N N N N N N N N N N N N N N N N N N N N N N N NNNNNNANNNNANNNNTTNNNNNNNNNNNNNNNGNNNTNNGNANNNNNNNANNNN NNNNNNN - 3'
4. Hasil sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primar ITS2
5' - NNNNNNNNNNNNNNNNNNTTTTGNTTCTTTTNNNTTTTGNTCNNAGCTNNNNNN N A C G T C C G C N N G G G G A C T A C A G A N N N N N N N N N N N N N N N T C C T C C G G C G G G C N C N N N N N T C C G G G G C T G C G A C G C A C C C G G G G C G T G A C C C C G C C N N N N N A A C A G T T T G G T AACGTTCACATTGGGTTTGGGAGTTGTAAACTCGGTAATGATCCCTCCGCTGGTTCA CCAACGGAGACCTTNNNNNNACTTTTACTNNNANNNNNNTNNNNNGATNCNNNNNN NTCTGNANTNANTTNNNNTANCNNATTNNNNNNGNGNNNNNCCNCNANGNNNAAAN CNNNNNNNNNNNNNNNNNAANCNTTTNNNNNNTTTTNNNTNNNNGNNANANNTNNN ANANNANGNCCNGNANNNNNANANTGNNNNNAGTTTGNTTGGTNNNNNCGNNGGG TCNCNNGGTNCNGGCGCTGCNANNNNNNNGCNCNNTNAANCCNNNAAATNNNNNA ANTTTNCNAANNGGNNNNTGNGGGGTTNGNNANTNNNNN - 3'
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Lampiran 5. Spektrogram sekuensing hasil PCR
1. Spektrogram sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer ITS5
ii«d ystems
E«kmMi_lrw(Hute 200« -0» -2e _ T N J e 3 _ ITSS_011 ln»l Mo<l«l/N«m« 3 1 0 < W 3 1 3 0 X L - 1 7 2 1 S - 0 a 9 T N J « 3 J T S 5 A u o 2 8 , 2 0 0 6 0 3 : 1 8 P M , I C T
S / N Q ; 5 9 A : 4 2 T : 3 6 C : 5 1 K B _ 3 1 3 0 _ P O P 4 _ B 0 T v 3 . m o b A u g 2 8 , 2 0 0 6 0 3 : 4 1 P M . I C T K B . b c p P l»2305 to l O S S S P k l L o o ; 2 3 0 S S p a o t n g : 1 2 . 8 PtWP«nel1500 K B 1.2 C»p:11 Ver»lOT 5 . 2 P 8 l c h 2 H I S Q V B«aa»: SOS P I f N « I T I « : s»qu«nc(ngL.2K)a06 M t l f M l f M H M » l l M i l M M N W C G M W S « »MC A T T .'-C C C O T T T . ^ C A c T .C CM IT MW W - G r G W»» K M M . ' : ' ' ^ A r T G T T It : : c T c O O C C G OO T C .^ 'C O C CC C O G G T G- G • G. K > . : : v : ; G C ,,"..:•,
9 n 25 33 41 49 57 S5 73 B I B9 97 l O S H 3
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1 3 1 12» 1 3 7 1 4 5 1 3 3 1 6 1 1 € 9 1 7 7 1 8 5 1 9 3 2 0 1 2 0 9 2 1 7 2 2 5 2 3 3 2 4 1
A T G A A O f i A C a c A G c a A . ' ^ A T G C G A t SA G T A A T o r G A f t t T G C A O.^ T C ^ G X O A - ^ T C.J-^T C G A ; , T C T X T G A A C G C ^ - C AT T G C G C c U GC C .^G T A T T c r G G C G G G C A r O C C T G 7 C C O A G C O 2 4 9 2 5 7 2 6 S 2 7 3 2 8 1 2«9 2 9 7 3 0 5 3 1 3 3 2 1 3 2 9 3 3 7 3 4 5 3 5 3 3«1 3«9
C C T C 6 c r •;• . G G G G Q : •: G G ' G T : G G G G - T ̂ G G O 'CC r z T : J ' t . r-.r G G G ' . G K ; C G L ' c :: C K - ' . . ' A T A , , : A G X G G : ' G G : " G • : G ' •••K • i : C : G : G - " - K . > :• r T G ' .:' ' ' : : : . » « • • :Ka G G 3 7 7 3 S S 3 9 3 4 0 1 4 0 9 4 1 7 4 2 5 4 3 3 4 4 1 4 4 9 4 5 7 4 S 5 4 7 3 4 6 1 4 8 9 4 9 7
C S C C G G . K G : . M » : a > - a M > K MM M G M . - G M M M M M • G : G . M M 1 ' . - I » M » : . M : H > M > H a K M G . M M M M M M
S O S 5 1 3 5 2 1 S 2 9 5 3 7 645 6 5 3 5 6 1 5 S 9 S 7 7 5 8 5 5 9 3 6 0 1 6 0 9
P r i n t a d o n : M o n A u g 2 8 , 2 0 0 4 0 S : 0 6 P M , I C T eiMciroph«ro8r*m D a t a P a g a 1 o f 1
3. spektrogram sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer rTS3
8 / N 0 :4«9 A ; 2 7 8 T ; 2 5 1 C : 3 4 T K B . b c p K B 1 . a C a p 4
El(Km»n_ln»titula_2006-12-13_TNJ«3-ITS3_004 T N J 6 3 - I T S 3
K B _ 3 1 3 0 _ P O P 4 _ B D T v 3 . m o b Pl» 2 7 0 7 to 1 0 6 8 5 P k l L o c : 2 7 0 7
V a r a h M i S.2 P a t c h S H S Q V B M M : 3 1 9
Ins t M o d a l / N a m e 3 1 0 0 / 3 1 3 0 X 1 - 1 7 2 1 6 - 0 2 9 D a c 1 3 . 2 0 0 6 0 4 : 0 9 P M . I C T D a c 1 3 . 2 0 0 6 0 4 : 3 2 P M . I C T
S p a c i n g ; 1 5 . 3 a P t B / P a n a l l S O O P l a t a N a n w : • • q _ 1 3 1 2 0 e
• m i l l • H M W i i ' i i i i c a M c i i • T T <mmi"s. T T C i i a T o » T < I I T c o ( A T C T T T G * ' C c c i ' c > r F a c G C C C V C C A Q T / ? T C T G 1 S C O O B C J T C C
1 « 33 34 45 56 t^ S9 100 i l l 122
n a m i A W i n i i i i T H H M MM M H X H M M I I M I I H r . x H H : M M C , H . R H H K H H H . H H H R N H H H M H H SOI S18 529 540 5S1
o n : T h u Oac 1 4 , 2 0 0 6 0 9 : 1 3 A M , I C T
• T C O O C e T T O G Q G f T C O GO »CC C C T C A C A C S O O T O C C G G C C C C S * A AT JC a G t G S C G G l C T C GC C O C .46 C C T C T C L - ' T G r GC. A G T AC T T T GcVc A A C ? C O C AC C 5 G O A ^ CAC O T C :; G i • I S 3 144 1S5 166 177 188 199 210 221 232 2 4 3
II d
E l B c t r o p h a r a g r a m D a t a P a g a 1 o f 1
Spektrogram sekuensing DNA Trichoderma sp. TNJ63 yang disekuens menggunakan primer 1TS2
mi G : 4 4 4 A:23a T : 3 0 8 C : 3 3 8 K B . b c p K B 1.2 C«p:2 « K M N M H W M M H K i m M M I M H T T T T 6»»T T C T T T T H H W T T T T GUT C»«C* O C T HH B H It « » AC GT C C S C H j a G G S J C I JlCA O A i m HHUMllH » H m t K I l l l T C C T C C O O C G G G CMC » H I W l l T C C G G O G C
1 2 2 3 34 4 5 5 6 «7 7 8 8 9 1 0 0 1 1 1
EVknfian_ln»tiluts_20Oe-12-13_TNJ63-ITS2_0oa T N J 6 3 - I T S 2
K B _ 3 1 3 0 _ P O F ' 4 _ B D T v 3 . m o b P\a 2772 t o 10585 P k l l.oc:2r?2
V a r a l c n 5 2 P a t c h 2 H I S Q V B a s e s : 2 S 7
I n s t Mod«l/N«m« 3 - 0 O . ' 3 i 3 O X L - 1 7 2 1 5 - 0 2 9 D a c 1 3 , 2 0 0 6 0 4 K ) 9 P M , ( C T D a c 1 3 , 2 0 0 6 0 4 : 3 2 P M , I C T
S ? a c i n o : i S 3 2 P t s / P a r . e l 1 SOO | g | a t c M a m a : »a<L.131209
r G C G AC O C A C C C G G G G C G T G A C C C C G CltM MHMA B C A G T T T GGT.AAC; G~ T C A C A T T O G O T T T G O G A G I T QV AA-AC T C G G I AA T G A T C C C T C C G C I OG T T C A C C A A C OG A G i C C T T MtHtJtll • A C T T T T A O 1 2 2 1 3 3 1 4 4 1 S 5 1 6 6 1 7 7 1 8 8 1 9 9 2 1 0 2 2 1 2 3 2 2 4 3
fit A A « « ,
i •WMMA MItM w m t m H I I 1 I W S . - T M C I M M M H W K T CT G H A » T W A M TTMMMMr A M CMMA T TMMWKWtOM GMMMMM c CM C M A M GMMBA A A « CMMMMMM MMMM M M M M W M M A A M C M T T T K M M M M M I T T T M M S T • «
2 5 4 2 6 5 27« 2 8 7 2 9 8 3 0 9 3 2 0 J 3 1 3 4 2 3 5 3 3 6 4 37l
MM G M MAMfM H T M M M A M A M MJM GWCCM3 M A MMMM M A V OMMMWMA G T T T GM T T S G TM MMM MCGMMGGG T CM CMMG GT T »CMGac;GCT OC M AMMMMMMMS (C MCMM'r »AAM C C » » » A A AlMMIfM MA i 3 8 6 3 9 7 4 0 8 US 4 3 0 4 4 1 4 5 2 4 6 3 4 7 4 4 8 S 4 9 6
M T T T M C M A A M MG CMMMMTGJGGOG- T M G tM A B T M *M MM S 0 7 5 1 8 5 2 9 5 4 0
P r t n t a c o n : T h u O a c 1 4 , 2 0 0 6 0 8 : 1 3 A M . I C T
Lampiran 6. Printout Gen Bank dari sekuens Trichoderma sp. TNJ63
LOCUS DEFINITION
ACCESSION VERSION KEYWORDS SOURCE
ORGANISM
REFERENCE AUTHORS
TI T L E JOURNAL
REFERENCE AUTHORS
TIT L E JOURNAL
FEATtJRES source
EF467659 632 bp DNA l i n e a r PLN 02-APR-2007 Trichoderma sp. TNJ63 18S r i b o s o m a l RNA gene, p a r t i a l sequence;
5.8S r i b o s o m a l RNA gene, and complete sequence; and 28S r i b o s o m a l
rRNA
raise RNA
rRNA
misc RNA
rRNA
i n t e r n a l t r a n s c r i b e d s p a c e r 1, i n t e r n a l t r a n s c r i b e d s p a c e r 2, RNA gene, p a r t i a l sequence. EF467659 EF467659.1 01:134143128
Trichoderma sp. TNJ63 Trichoderma sp. TNJ63 Eulcaryota; Fungi; Ascomycota; Pezizomycotina; S o r d a r i o m y c e t e s ; Hypocreomycetidae; H y p o c r e a l e s ; m i t o s p o r i c H y p o c r e a l e s ; Trichoderma. 1 (bases 1 to 632) Nugroho,T.T., R e s t u h a d i , F . , Ito.T.R., F a i s a l , F . , Saryono,S. and C h a i n u l f i f f a h , A . M . M o l e c u l a r i d e n t i f i c a t i o n of R i a u b i o c o n t r o l Trichoderma s t r a i n s Unpublished 2 (bases 1 t o 632) Nugroho,T.T., R e s t u h a d i , F . , Ito,T.R., F a i s a l , F . , Saryono,S. and C h a i n u l f i f fah, A. M. D i r e c t Submission Submitted (02-MAR-2007) Department of Chemistry, FMIPA, U n i v e r s i t y of R i a u , Kampus Binawidya J l . Raya Soebrantas IQn 12,5, PeJcanbaru, R i a u 28293, I n d o n e s i a
L o c a t i o n / Q u a l i f i e r s 1..632 /organism="Trichodenna sp. TNJ63" /mol_type="genomic DNA" / s t r a i n = " T N J 6 3 " / i s o l a t i o n _ s o \ u r c e = " R i a u c i t r u s grove s o i l " /db_xref="taxon:434037" / c o u n t r y = " I n d o n e s i a " /note="PCR_j)rimers=fwd_name: I T S 5 , rev_name; I T S 4 ; PCR_j5rimers=fwd_name: I T S 3 , rev_name: I T S 2 " <1..60 /product="18S r i b o s o m a l RNA" 61..242 / p r o d u c t = " i n t e r n a l t r a n s c r i b e d s p a c e r 1" 243..400 /product="5.8S ribosomal RNA" 401..574 / p r o d u c t = " i n t e r n a l t r a n s c r i b e d s p a c e r 2" 575..>632 /product="28S r i b o s o m a l RNA"
ORIGIN
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1 t t c t t g g a a g t a a a a g t t c g t a a c a a g g t c t c c g t t g g t g aaccagcgga g g g a t c a t t a 61 c c g a g t t t a c a a c t c c c a a a c c c a a t g t g a a c g t t a c c a a a c t g t t g c c t cggcggggtc
121 acgccccggg t g c g t c g c a g c c c c g g a a c c aggcgcccgc cggaggaacc a a c c a a a c t c 181 t t t c t g t a g t c c c c t c g c g g a c g t a t t t c t t a c a g c t c t g a g c a a a a a t t c a a a a t g a a t 241 c a a a a c t t t c aacaacggat c t c t t g g t t c t g g c a t c g a t gaagaacgca gcgaaatgcg 301 a t a a g t a a t g t g a a t t g c a g a a t t c a g t g a a t c a t c g a a t c t t t g a a c g c a c a t t g c g c c 361 c g c c a g t a t t ctggcgggca t g c c t g t c c g a g c g t c a t t t c a a c c c t c g a a c c c c t c c g g 421 gggatcggcg ttg g g g a t c g g g a c c c c t c a cacgggtgcc ggccccgaaa t a c a g t g g c g 481 g t c t c g c c g c a g c c t c t c c t gcgcagtagt t t g c a c a a c t cgcaccggga gcgcggcgcg 541 t c c a c g t c c g t a a a a c a c c c a a c t t t c t g a a a t g t t g a c c tcggatcagg t a g g a a t a c c 601 c g c t g a a c t t a t c c t t t t c t a t t t t c c g g a ag
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