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8/18/2019 Cloning Into Plasmids -Riboprobes
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Cloning intoPlasmids
Restriction Fragment Cloning & PCR
Cloning by the Topo TA™ Method
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Cloning Vectors
The molecular analysis of DNA has been made possible by thecloning of DNA The t!o molecules that are re"uired for cloning arethe DNA to be cloned and a cloning #ector
Cloning vector $ a DNA molecule that carries foreign DNA into ahost cell% replicates inside a bacterial or yeast' cell and producesmany copies of itself and the foreign DNA
Three features of all cloning vectors se"uences that permit the propagation of itself in bacteria or in yeast
for (ACs' a cloning site to insert foreign DNA) the most #ersatile #ectors
contain a site that can be cut by many restriction en*ymes a method of selecting for bacteria or yeast for (ACs' containing a
#ector !ith foreign DNA) uually accomplished by selectable mar+ersfor drug resistance
8/18/2019 Cloning Into Plasmids -Riboprobes
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Types of Cloning Vectors
Plasmid $ an e,trachromosomal circular DNA molecule thatautonomously replicates inside the bacterial cell) cloning limit- .//to ./%/// base pairs or /.$./ +ilobases +b'
Phage $ deri#ati#es of bacteriophage lambda) linear DNA
molecules% !hose region can be replaced !ith foreign DNA!ithout disrupting its life cycle) cloning limit- 0$1/ +b
Cosmids $ an e,trachromosomal circular DNA molecule thatcombines features of plasmids and phage) cloning limit $ 23$3/ +b
Bacterial Artificial Chromosomes (BAC) $ based on bacterialmini$F plasmids cloning limit- 43$2// +b
Yeast Artificial Chromosomes (YAC) $ an artificial chromosomethat contains telomeres% origin of replication% a yeast centromere%and a selectable mar+er for identification in yeast cells) cloninglimit- .//$./// +b
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General Steps of Cloning with Any Vector prepare the #ector and DNA to be cloned by
digestion !ith restriction en*ymes to generate
complementary ends e,ception Topo cloning see
later slides' ligate the foreign DNA into the #ector !ith the
en*yme DNA ligase
introduce the DNA into bacterial cells or yeast cells
for (ACs' by transformation select cells containing foreign DNA by screening for
selectable mar+ers usually drug resistance'
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Restriction & Ligation
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Insertion of RestrictionFragment into Vector
Multi Cloning Site
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The pC !" or !# Cloning
Vector
R5 6ites in blue occur only once in the plasmid
Lac Z α
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Transformation andSelection
6creening
hite hite Blue !ead
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The Topo TA PCR Cloning Vector
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Features of Topo Vector
EcoR 7 sites flan+ing the PCR product insertion site
for easy remo#al of inserts
8anamycin and ampicillin resistance genes for your
choice of selection in E. coli 5asy blue9!hite screening of recombinant colonies
Promoter9priming sites for in vitro transcription
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The Topo TA CloningProcess
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$hy Are $e Topo Cloning%
PCR generates the e,act gene fragment !e !ant toclone
PCR products and no other DNA are ligated into the
Topo #ector by the topoisomerase
:igation is highly efficent as high as ;/
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Generating Rioproes
T4 =acteriophage RNA polymerase can be used to transcribe the
insert in the left!ard direction to ma+e single stranded RNA The
strand !hich is copied template strand' depends on the orientation of
the insert >e !ill need to restriction map our plasmid s to determine
the orientation of the insert 6ince insertion is random =othorientations should be represented in our clone population >e need
to select plasmids !hich !ill generate RNA !hich is complimentary to
the mRNA !e are attempting to locali*e by in situ hybridi*ation