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26/02/2014

DNA RECOMBINANT TECHNOLOGY

Agustina Setiawati Fakultas Farmasi USD

Bioteknologi 2011

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REFERENCE

Bioteknologi 2011

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26/02/2014 DEFINISI DNA Cloning – the act of making many identical copies of a particular piece of DNA (often a gene) As you know. the first stop often involves joining a piece of DNA of interest to a cloning vector using DNA ligase Bioteknologi 2011 3 VECTORVECTOR -Plasmid Plasmid is extrachromosome D A with s!ecial characteristic" 1# 2# 3# 4# '# F !lasmid $ !lasmid %ol !lasmid &irulence !lasmid Degradati(e !lasmid Does eucar)ote ha(e !lasmid* Bioteknologi 2011 4 2 .

)me +as !romoter and $BS %ouldn/t 0e trans-erred to one or more s!ecies Bioteknologi 2011 1 3 .26/02/2014 VECTORVECTOR .nl) one restriction site -or se(eral endonuclease en.Plasmid Bioteknologi 2011 ' Plasmid to be a Clo i ! Ve"to# Small +a(e s!ecial marker .

26/02/2014 Plasmid b#i !s a tibioti" #esista "e !e e Bioteknologi 2011 2 VE$TORVE$TOR .!hage 4n-ection l)sis5c)cle o.Ba%te#io&a!e 3ain structure o.Phage Bioteknologi 2011 6 4 .

26/02/2014 8)sogenic c)cle Bioteknologi 2011 7 Plasmid as Ve"to#' ()C* + (BR.-- Bioteknologi 2011 10 5 .

26/02/2014 Clo i ! P#i "i(al CLONING STEP 1# 2# 3# 4# '# Pre!are D A target and s!esi-ic (ector %ut D A target and (ector using retriction endonuclease 8igate them 9rans-orm into li(ing cells Selection Bioteknologi 2011 12 6 .

A Hpa4 7 ./ BL)NT ENDS P:3.9.26/02/2014 ./ C)T DNA a d Ve"to# 0si ! e do 0"lease Bioteknologi 2011 13 . .

.9.26/02/2014 -/ Sti"%1 E ds P:3.A Eco$4 8 .

26/02/2014 -/ Li!atio DNA ta#!et + 2e"to# Stick) ends has higher e--icienc) due to <catch hold= each other Blunt ends need higher D A concentration to ligate one another +ow to increase ligation e--icienc) o0lunt ends* Bioteknologi 2011 12 St#ate!1' ./ (0t sti"%1 e d i to bl0 t e d 3ea% ess4 Bioteknologi 2011 16 9 .

26/02/2014 St#ate!1' -/ P0t a ada(to# An ada!tor has stick) ends linker and could ligate one to another Bioteknologi 2011 17 5/ T#a s&o#matio i Li2i ! Cells Bioteknologi 2011 20 10 .

26/02/2014 T#a s&o#matio i a imal + (la t "ells Bioteknologi 2011 21 P61si"al Met6od to t#a s&o#m #e" DNA Bioteknologi 2011 22 11 .

enetic ma!!ing o.!U%6 Bioteknologi 2011 23 $eaction in :#coli ha(ing !U%6 12 .26/02/2014 7/ SELECTION-PLASMID ()C* VECTOR .

26/02/2014 IPTG: isopropyl thiogalactoside 13 .

VECTOR .-.!Br322 Bioteknologi 2011 22 Sele"tio Bioteknologi 2011 26 14 .26/02/2014 7/ SELECTION-PLASMID (B#.enetic ma!!ing o.

26/02/2014 Bi a#1 Clo i ! s1stem :#%oli !lasmid has homolog) in 9i5!lasmid so the) can 0e integrated 9he) trans-ormed to A#tume-acien that a0le to in-ect !lant cells Bioteknologi 2011 27 T#a s&o#matio #e"ombi a t (lasmid i (la t Bioteknologi 2011 30 15 .

26/02/2014 S)CCESS PROBABILTY %alculated 0) %8A$%> ? %A$B.success A7'B or 77BC a " inserted genom 0 " total genom in s!ecies P#oblem4 Bioteknologi 2011 32 16 . e@uation P" !ro0a0ilit) o.

26/02/2014 Bioteknologi 2011 33 17 .