1. Miniprep
2016.1학기 기초유전학실험

2. 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.)

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

4. Transformation
Mini prep
Ligastion된 DNA

5. D-1
Inoculation

6. Transformed E.coli
Chromosomal DNA
Plasmid DNA

7. Boiling method
(Genomic DNA)
(Plasmid DNA)

8. Alkaline-lysis method

9. Ethanol precipitation (Ethanol p.p.t)

10. 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.

11. 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