Digestion 2016 년 1 학기 기초유전학실험. Structure of vector.

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Presentation transcript:

Digestion 2016 년 1 학기 기초유전학실험

Structure of vector

pMB bp promoter luciferase Amp R Ori BamHI EcoRI Ori : replication origin - 재조합 DNA 의 증폭가능 Amp : antibiotic resistant gene – 항생제 저항 유전자 Promoter : 유전자에서 단백질로 발현이 시작되는 부위 ( 스위치 역할 ) Luciferase (reporter gene) : promoter 활성을 측정하기 위한 척도 MCS (multiple cloning site) : restriction enzyme site (ex. BamHI, EcoRI, etc.)

Subcloning

1.Digestion  today 2.Ligation 3.Transformation 4.Mini-prep of plasmid DNA 5.Screening for the correct clone Purification 6. Purification of cloned DNA with sillica oxide (midi-prep) DNA transfer to cells 7. Transient transfection of cloned DNA into human cells 8. Assaying gene expression in the tranfected cells

Digestion Ligation Cut! (Restriction Enzyme) Ligation (ligase)

Transformation Mini prep

Screening Midi prep 틀림 ? 맞음 ? Mini prep 된 DNA

Transfection Assaying

-GAATTC- -CTTAAG- -GGATCC- -CCTAGG- CUT!

Nucleotide Nucleotide= Phosphate group + Sugar + Base(A,T,G,C) Phospho- diester - bond Hydrogen- bond

pMB bp promoter luciferase Amp R Ori BamHI EcoRI