1. Digestion 2016 년 1 학기 기초유전학실험

2. Structure of vector

3. pMB01 5747bp 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.)

4. Subcloning

5. 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

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

7. Transformation Mini prep

8. Screening Midi prep 틀림 ? 맞음 ? Mini prep 된 DNA

9. Transfection Assaying

10. -GAATTC- -CTTAAG- -GGATCC- -CCTAGG- CUT!

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

13. pMB01 5747bp promoter luciferase Amp R Ori BamHI EcoRI