유전자조작기법실험

목차

실험목표

실험개요
I. PCR
II. Construction of Recombinant DNA
III. Transformation
IV. Characterization of Recombinants

실험내용
1. PCR of λDNA with LP primers & Gel Electrophoresis
2. Purification of the PCR Product (Amplified from λ DNA)
3. GE of the PCR Product & the Purified PCR Product
4. RE-digestion of the Purified PCR Product and a Cloning Vector
5. Ligation of the Digested DNAs
6. Preparation of Competent Cells & Transformation
7. Culture of Recombinants & Miniprep of Plasmid
8. RE-Analysis of the Isolated Plasmid

본문내용

d 900㎕ pre-warmed LB medium
↓
Incubate at 37℃ for 1.5hr, with shaking at 150rpm
↓
spread 100㎕ on LB plate(X-gal, Amp)
↓
Incubate at 37℃ O/N
↓
Colony Selection
- cells carrying a recombinant DNA : white colony
- cells carrying a self-ligated vector : blue colony
7. Culture of Recombinants & Miniprep of Plasmid
(1) cell culture
LB (+Amp) broth에 single white/blue colony 각 1개씩을 선택해서 inoculation
↓
Incubate at 37℃, with shaking at 200rpm, for O/N
(2) Preparation of plasmid DNA

Transfer 1㎖ cell suspension to 1.5㎖ tube
↓ cfg at max speed, for 20"
↓ drain supernatant

add 500㎕ ice-cold STE (sol 0)
↓
tap and vortex for 5"
↓ cfg at max speed, for 20"
↓ remove supernatant

add 100㎕ ice-cold GTE (sol I)
↓ pipet completely
↓ at RT for 5'
DNA Denaturation>
add 200㎕ SDS/NaOH (sol II)
↓ invert 5 times (pipetting-vortexing)
↓ on ice for 5'
and Precipitation>
add 150㎕ ice-cold KOAc/HOAc (sol III)
↓ invert 5 times
↓ incubate on ice for 5'
with plasmid DNA>
↓ cfg at max speed, for 5'
Transfer supernatant to new tube(≒400㎕)
↓
and Harvest>
add 1V isopropanol (400㎕)
↓ invert 5 times
↓ at RT for 2' exactly
↓ cfg at max speed, for 5'
↓ remove supernatant
& Reharvest>
add 250㎕ ice-cold 95% EtOH
↓ vortex for 5", and cfg at max speed for 5'
↓ remove supernatant
＜Drying of DNA pellet＞
dry DNA pellet
↓
＜Dissolving DNA＞
dissolve DNA with 20㎕ TE (+RNase 0.5㎕)
8. RE-Analysis of the Isolated Plasmid
white colony
blue colony
DNA
10X Buffer
Bam HI
Hind III
dH2O
5㎕
1㎕
0.25㎕
0.25㎕
3.5㎕
5㎕
1㎕
0.25㎕
0.25㎕
3.5㎕
Total
10㎕
10㎕
→ incubate at 37℃ for 30'
→ GE on 1.0% agarose gel(2.7kb/0.8kb)
→ EtBr staining & viewing, photography
< A map of pGEM-3Z Vector >

키워드

유전자,   유전자조작,   유전자조작기법,   임상병리,   유전자 조작기법 실험,   병리학실험