Amplifikasi dan Kloning Gen Protein Disulfida Isomerase Ragi Menggunakan Vektor T  

Oleh:

Purkan

Jenis: Article from Journal - ilmiah nasional
Dalam koleksi: Jurnal Penelitian Medika Eksakta vol. 04 no. 03 (Dec. 2003), page 231-236.
Topik: Protein disulfide isomerase; amplifiying; PCR; cloning; Saccharomyces cerevisiae
Fulltext: Purkan.pdf (153.44KB)

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Perpustakaan PKPM Nomor Panggil: J101 Non-tandon: 1 (dapat dipinjam: 0) Tandon: tidak ada

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Protein disulfide isomerase (PDI, E.C.5.3.4.1) encoded by PDI1 gene of yeast is an enzyme that catalyst the formation and arrangement of disulfide bond on process of polypeptide folding. Amplifying and cloning of yeast PDI1 gene with T vector was done as preliminary research on over expression of the gene in E. coli. The aims of the research were to amplify the protein disulfide isomerase gene by PCR, to state the size of gene resulted by PCR, to obtain pGemT-PDI1 recombinant in a clone of E. coli DH5 a. Amplification of yeast PDI1 gene with PCR was run by using two primers, namely forward and reverse primers. Then, the amplicon that resulted by PCR was ligated with T vector (pGemT) and used to transform E. coli DH5 a. Selection of transformans were done to obtain a clone of E. coli DH5 a that contain pGemT-PDI1 recombinant. DNA fragment size of ± 1700 bp that equivalence with the size of PDI1 gene have been obtained from amplification of yeast PDI1 gene with PCR. A DNA fragment ( ± 4700 bp) was resulted by digestion of pGemT-PDI1 recombinant with single enzyme, NdeI, and BamHI, and two DNA fragment ( ± 3000 bp) and ( ± 1700 bp) with both enzymes showed that PDI1 gene have been contructed and cloned in E. coli DH5 a. Suggestions of this research are : it will be important to sequence nucleotides of PDI1 gene that be cloned successfully.

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