Kloning gen coat protein smv dengan pendekatan pcr: (Cloning of the coat protein gene of smv with the pcr approach )
ABSTRACT SMV is RNA virus had to be converted to the first strand DNA using oligo (dT) and murine reverse transcriptase. Amplification of coat protein gene region was carried out by Polimerase Chain Reaction (PCR) with two primers. The PCR product was blund ended by SI nuclease, and ligated into Sma...
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| Format: | Article NonPeerReviewed |
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[Yogyakarta] : Universitas Gadjah Mada
1996
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| Online Access: | https://repository.ugm.ac.id/24780/ http://i-lib.ugm.ac.id/jurnal/download.php?dataId=7758 |
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| Institution: | Universitas Gadjah Mada |
| Summary: | ABSTRACT
SMV is RNA virus had to be converted to the first strand DNA using oligo (dT) and murine reverse transcriptase. Amplification of coat protein gene region was carried out by Polimerase Chain Reaction (PCR) with two primers. The PCR product was blund ended by SI nuclease, and ligated into Smal digested pUC 18 and phosphatase treatment by calf intestine phosphatase. Ligation mixture was used to transform E. coli DHS a Recombinant plasmid was digested with EcoRland HindIII showed 0,8 kb fragment. Southern blot analysis at high strigency using PCR product as a probe shows that the 0,8 kb fragment produced intense signal.
Key words : SMV, coat protein IN'I1SARI
SMV merupakan virus RNA sehingga sebagai langkah pertama dalam penelitian ini perlu dilakukan sintesis untai pertama cDNA dengan menggunakan primer oligo-(dT) dan murine reverse Iranscriptase. Kemudian amplifikasi daerah gen coat protein dilakukan dengan metode Polimerase Chain Reaction (PCR) dengan menggunakanuntas pertama cDNA sebagai cetak awal dan duo primer yang terdiri d ari 5'-TACATCTTGGAACCAATGGCAGGCAAGGAGAGAAG-3' dan 5'-AGGACAACAAACATTGCCG-3'. Hasil PCR dibuat papak dengan nuklease s I, diligasikan dengan pliCI8 yang telah dipotong dengan Smal dan dihilangkan ujung fosfatnya dengan calf intestine uhosphatase (CIP). Transforrnasi dilakukan pada E. coli DH5a. Plasmid rekombinan yang didigesti �dengan EcoRI dan Hind11.1 menunjukkan fragmen 0,8 kb. Analisis Southern blot pada suhu tinggi dengan pelacak flagmen hasil PCR menunjukkan fragmen tersebut memberikan signal kuat.
Kam kunci : SMV, coat protein |
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