Konstruksi gen penyandi pluripotensi c-Myc dan Sox2 rekombinan di inang Escherichia coli = Construction of recombinant gene coding of pluripotency c-Myc and Sox2 in Escherichia coli host
| Main Authors: | Tampubolon, Tiodinar Theresia, author, Add author: Amarila Malik, supervisor, Add author: Septelia Inawati Wanandi, examiner, Add author: Maksum Radji, examiner, Add author: Arry Yanuar, examiner |
|---|---|
| Format: | Bachelors Thesis |
| Terbitan: |
Universitas Indonesia
, 2014
|
| Subjects: | |
| Online Access: |
http://lontar.ui.ac.id/detail?id=20439707 |
| ctrlnum |
20439707 |
|---|---|
| fullrecord |
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<dc schemaLocation="http://www.openarchives.org/OAI/2.0/oai_dc/ http://www.openarchives.org/OAI/2.0/oai_dc.xsd"><type>Thesis:Bachelors</type><title>Konstruksi gen penyandi pluripotensi c-Myc dan Sox2 rekombinan di inang Escherichia coli = Construction of recombinant gene coding of pluripotency c-Myc and Sox2 in Escherichia coli host</title><creator>Tampubolon, Tiodinar Theresia, author</creator><creator>Add author: Amarila Malik, supervisor</creator><creator>Add author: Septelia Inawati Wanandi, examiner</creator><creator>Add author: Maksum Radji, examiner</creator><creator>Add author: Arry Yanuar, examiner</creator><publisher>Universitas Indonesia</publisher><date>2014</date><subject>Cloning</subject><subject>Cloning in literature</subject><subject>Cloning, Molecular -- Methods -- Experiments</subject><subject/><description>Peningkatan ekspresi gen Sox2 dan c-Myc telah dilaporkan memiliki korelasi dengan tingkat keparahan kanker payudara. Sox2 dan c-Myc merupakan faktor transkripsi utama yang berperan dalam proses diferensiasi sel punca kanker. Penelitian ini bertujuan mengkonstruksi gen penyandi pluripotensi Sox2 dan c-Myc ke dalam sel inang Escherichia coli DH5&#945; dengan menggunakan plasmid pET101/D-TOPO. Prinsip kloning yang dilakukan adalah dengan mengklon DNA binding domain dari gen Sox2 dan c-Myc. Untuk mendapatkan DNA Sox2 dan c-Myc, dilakukan reverse-transcriptase polymaerase chain reaction (RT-PCR) dengan menggunakan template mRNA dari sel punca kanker payudara serta aplifikasi dengan PCR untuk mendapatkan fragmen DNA dalam jumlah banyak. Forward primer dan reverse primer yang digunakan dirancang dengan menggunakan data dari NCBI GenBank dan UNIPROT serta program Serial Cloner dan PerlPrimer. Sebelum dikloning, dilakukan sekuensing. Hasil sekuensing dianalisis dengan menggunakan BLAST. Fragmen DNA diligasi berdasarkan prinsip penambahan empat basa pada forward primer (CACC) yang overhang terhadap ujung 5' vektor kloning (GTGG). Hasil ligasi ditransformasi menggunakan metode secara kimia dengan CaCl2 dan heat shock. Koloni yang tumbuh direplika dan dilakukan isolasi plasmid. Hasil PCR plasmid rekombinan menunjukkan bahwa gen Sox2 dan c-Myc berhasil disisipkan ke dalam vektor.
<hr>
Increase in Sox2 and c-Myc gene expression have been reported to correlate with the severity of breast cancer. Sox2 and c-Myc is the major transcription factor in the process of stem cell differentiation. The objective of this study is to construct the gene coding of pluripotency, Sox2 and c-Myc, into Escherichia coli DH5&#945; host cell by using the pET101/D-TOPO vector. The cloning principle is to clone the binding site domain of Sox2 and c-Myc. In order to get the Sox2 and c-Myc DNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out using mRNA template from breast cancer stem cell and amplification using PCR to obtain DNA fragment in large quantities. Forward primer used were designed using the data from NCBI GenBank and UNIPROT with Serial Cloner and PerlPimer program. Sequencing was carried out before the cloning process. The sequencing result were analyzed using BLAST. DNA frgament was ligated using principle of four base addition to the forward primer (CACC) which overhang in the 5' and of cloning vector (GTGG). The ligation product was transformed using the chemical method with CaCl2 and heat shock. The colonies were replicated and the plasmid was isolated. The result showed that Sox2 and c-Myc was successfully inserted into the vector.</description><identifier>http://lontar.ui.ac.id/detail?id=20439707</identifier><recordID>20439707</recordID></dc>
|
| format |
Thesis:Bachelors Thesis Thesis:Thesis |
| author |
Tampubolon, Tiodinar Theresia, author Add author: Amarila Malik, supervisor Add author: Septelia Inawati Wanandi, examiner Add author: Maksum Radji, examiner Add author: Arry Yanuar, examiner |
| title |
Konstruksi gen penyandi pluripotensi c-Myc dan Sox2 rekombinan di inang Escherichia coli = Construction of recombinant gene coding of pluripotency c-Myc and Sox2 in Escherichia coli host |
| publisher |
Universitas Indonesia |
| publishDate |
2014 |
| topic |
Cloning Cloning in literature Molecular -- Methods -- Experiments |
| url |
http://lontar.ui.ac.id/detail?id=20439707 |
| contents |
Peningkatan ekspresi gen Sox2 dan c-Myc telah dilaporkan memiliki korelasi dengan tingkat keparahan kanker payudara. Sox2 dan c-Myc merupakan faktor transkripsi utama yang berperan dalam proses diferensiasi sel punca kanker. Penelitian ini bertujuan mengkonstruksi gen penyandi pluripotensi Sox2 dan c-Myc ke dalam sel inang Escherichia coli DH5α dengan menggunakan plasmid pET101/D-TOPO. Prinsip kloning yang dilakukan adalah dengan mengklon DNA binding domain dari gen Sox2 dan c-Myc. Untuk mendapatkan DNA Sox2 dan c-Myc, dilakukan reverse-transcriptase polymaerase chain reaction (RT-PCR) dengan menggunakan template mRNA dari sel punca kanker payudara serta aplifikasi dengan PCR untuk mendapatkan fragmen DNA dalam jumlah banyak. Forward primer dan reverse primer yang digunakan dirancang dengan menggunakan data dari NCBI GenBank dan UNIPROT serta program Serial Cloner dan PerlPrimer. Sebelum dikloning, dilakukan sekuensing. Hasil sekuensing dianalisis dengan menggunakan BLAST. Fragmen DNA diligasi berdasarkan prinsip penambahan empat basa pada forward primer (CACC) yang overhang terhadap ujung 5' vektor kloning (GTGG). Hasil ligasi ditransformasi menggunakan metode secara kimia dengan CaCl2 dan heat shock. Koloni yang tumbuh direplika dan dilakukan isolasi plasmid. Hasil PCR plasmid rekombinan menunjukkan bahwa gen Sox2 dan c-Myc berhasil disisipkan ke dalam vektor.
<hr>
Increase in Sox2 and c-Myc gene expression have been reported to correlate with the severity of breast cancer. Sox2 and c-Myc is the major transcription factor in the process of stem cell differentiation. The objective of this study is to construct the gene coding of pluripotency, Sox2 and c-Myc, into Escherichia coli DH5α host cell by using the pET101/D-TOPO vector. The cloning principle is to clone the binding site domain of Sox2 and c-Myc. In order to get the Sox2 and c-Myc DNA, reverse-transcriptase polymerase chain reaction (RT-PCR) was carried out using mRNA template from breast cancer stem cell and amplification using PCR to obtain DNA fragment in large quantities. Forward primer used were designed using the data from NCBI GenBank and UNIPROT with Serial Cloner and PerlPimer program. Sequencing was carried out before the cloning process. The sequencing result were analyzed using BLAST. DNA frgament was ligated using principle of four base addition to the forward primer (CACC) which overhang in the 5' and of cloning vector (GTGG). The ligation product was transformed using the chemical method with CaCl2 and heat shock. The colonies were replicated and the plasmid was isolated. The result showed that Sox2 and c-Myc was successfully inserted into the vector. |
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Universitas Indonesia |
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