cloning into plasmids -riboprobes

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


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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

cloning into plasmids -riboprobes

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