Miniprep 2016.1학기 기초유전학실험.

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Presentation transcript:

Miniprep 2016.1학기 기초유전학실험

promoter luciferase Ori Amp R Ori : replication origin - 재조합 DNA의 증폭가능 pMB01 5747bp promoter luciferase Amp R Ori BamHI EcoRI Ori : replication origin - 재조합 DNA의 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.)

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

Transformation Mini prep Ligastion된 DNA

D-1 Inoculation

Transformed E.coli Chromosomal DNA Plasmid DNA

Boiling method (Genomic DNA) (Plasmid DNA)

Alkaline-lysis method

Ethanol precipitation (Ethanol p.p.t)

7.4 Procedure (D-day) transfer bacterial suspensions to clean microfuge tubes (label the tubes ) spin down at maximal speed for 1 minute using a microfuge (BALANCE!!) discard the supernatant (remove as much as possible) add 200 ul of the lysis buffer and resuspend by vortexing heat the tubes for 40 seconds (temperature should be over 95oC), 5min incubation at room temperature spin down at maximal speed for 10 minutes using a microfuge prepare tubes by adding 2 ul of 5M NaCl to each of clean tubes (labeling) transfer the supernatant to NaCl-containing tubes by decanting mix well, add 250 ul of isopropanol to each of the tubes, and mix well again spin down at maximal speed for 15 minutes using a microfuge - microfuge tube 뚜껑과 몸체의 연결부위가 밖을 향하도록 넣는다.

discard the supernatant add gently 400 ul of 70% ethanol to each tube, close the cap, and roll gently to wash the inner surface of the tube add gently 400 ul of 100% ethanol to each tube, close the cap, and roll gently to wash the inner surface of the tube remove ethanol completely dry the pellet (air-dry or use 65oC temp-block or incubator) - the pellet should be completely dry resuspend DNA with 20 ul of DDW mix 1 ul of miniprep DNA with 5 ul of loading buffer and load onto an agarose gel gel electrophoresis (below 100V) for about 20 minutes What do you see in the gel ? check for the plasmid DNA band store minipreped DNA frozen