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DNA Activation
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Descrição: the new generations
DNA REKOMBINAN
Cloning Vectors
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Apakah teknologi DNA Rekombinan? Pembentukan kombinasi materi genetik yang baru dengan penyisipan molekul DNA ke dalam suatu vektor sehingga terintegrasi dan mengalami
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A N D n a n i b m o k e R n
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Tahapan DNA Rekombinan 1. Isolasi DNA 2. Pemo Pemoto tong ngan an & pen pengg ggab abun unga gan n mole moleku kull DNA DNA 3. Kloning DNA a. Pe Peny nyis isip ipan an fr frag agme men n DNA DNA ke da dala lam m vek vektor tor b. Vektor kloning
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SUB-KLONING (Insersi gen pada vektor ): teknik untuk memindahkan gen interes dari par dari parent ent vector ke destination vector Enzim restriksi : memotong gen interes (insert (insert ) dari parental. •
Insert dimurnikan dengan isolasi (metode :gel : gel isolation) isolation)
•
Gen digandakan dengan PCR
•
Enzim Restriksi yang sama : untuk mendigesti gen tujuan Penggunakan enzim yang sama agar complementary sticky n aends p phosphatase ( (Calf Calf Intestinal Alkaline Phosphatase; Phosphatase ; CIAP) a phosphatase hdigunakan untuk mencegah ligasi sendiri a •
•
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VEKTOR KLONING Bagian DNA dimana fragmen DNA asing dapat disisipkan •
Digunakan untuk menggandakan molekul DNA •
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KARAKTERISTIK VEKTOR 1. Memiliki or i gi n of r epl i cation sehingga DNA dapat digandakan dalam sel inang 2
Mudah diisolasi dan tidak terdegradasi selama pemurnian
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VEKTOR KLONING A. Vektor Bakteri 1.
Plasmid
2.
Bakteriofag
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VEKTOR KLONING-PLASMID
Bagian Ekstrakromosom DNA : tidak terlalu dibutuhkan bakteri Memiliki beberapa fungsi : resisten antibiotik, menghasilkan pigmen, degradasi komponen dan fiksasi dinitrogen Dapat disisipi DNA sampai 10 kb
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Plasmid-Good Cloning Vectors •
Ukuran kecil (mudah untuk manipulasi dan isolasi)
•
Sirkuler (lebih stabil)
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PLASMID
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Penyisisipan DNA asing pada Plasmid
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Kerugian menggunakan plasmid
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BACTERIOPHAGE
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Konfirmasi Keberadaan Insert Untuk
memastikan pertumbuhan bakteri hanya bakteri transforman (pembawa plasmid diinginkan)
Gen
penanda digunakan dalam vektor untuk seleksi. Gen penanda khas khas resistensi antibiotik atau biosintesis biosintesis nutrisi. Contoh,
"penanda gen" bisa resistensi terhadap antibiotik :ampicillin. Jika
bakteri mengandung plasmid dengan gen interest maka mereka juga akan berisi "gen penanda".
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Konfirmasi Keberadaan Insert
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Jawablah beberapa pertanyaan berikut ini! 1.
Apak Ap akah ah te tekn knol olog ogii DN DNA A rek rekom ombi bina nan? n?
2.
Baga Ba gaim iman anaa tah tahap apan an su subb-kl klon onin ing? g?
3.
Apaa yan Ap yang g dim dimak aksu sud d den denga gan n ve vekt ktor or kl klon onin ing g?
4.
Apaa sa Ap saja ja ka kara rak kte teri rist stiik vek ekto torr?
5.
Sebu Se butk tkaan mac acaam ve vekt ktor or kl klon onin ing! g!
6.
Vekt Ve ktor or klo kloni ning ng apa apa yan yang g dapa dapatt digu diguna naka kan n untu untuk k sel sel euka eukary ryot otik ik
7.
Apaa keunt Ap keuntun unga gan n dan dan kerugi kerugian an men mengg ggun unak akan an pla plasm smid id seba sebaga gaii vekt vektor or??
8.
Apa fungsi selectable marker marker , uni un i que r estr i cti ction on si si te tes s, dan origin of replication?
9.
Dalam me Dalam mengi nginfe nfeksi ksi sel bak bakter teri, i, bak bakter teriof iofag ag me memi milik likii 2 jalu jalur, r, seb sebutk utkan, an, jelaskan dan gambarkan secara secara skematis! skematis!
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