목차
1. 제목
2. 이름
3. 실험 목적
4. 실험 방법
5. 결과
6. 고찰
7. reference
본문내용
1. 제목 : 세포 형질 전환
3. 실험 목적
Plasmid DNA 증식을 위해 E.coli에 transformation 후, 293t cell에 transfection을 시킴으로써 Plasmid DNA를 진핵세포에 도입하기 위한 실험이다.
4. 실험 방법
4-1. transformation
1) competent cell(20㎕)에 plasmid GFP DNA(1/10, 2㎕) 넣고 ice에서 5분
2) 42℃, 45초
3) ice에서 식히기
4) 100㎕ LB liquid 배지(S.O.C) 추가하기
5) amp + LB 배지에 spreading 후 뒤집어서 37℃ 배양
6) 다음날 colony 확인 및 파라필름 감싸서 4℃ 보관
4-2. plasmid DNA prep
1) 50ml conical tube에 배양한 E.coli를 4000rpm, 20분 centrifuge
2) 상층액 제거 후 A1 buffer 150㎕ 넣고 vortexing, 1.7ml EP tube에 옮김
3) A2 buffer 250㎕ 넣고 inverting 후 RT에서 2분 incubation
4) A3 buffer 350㎕ 넣고 파란색이 사라질 때 까지 inverting 후 centrifuge 17000rpm, 3분
참고 자료
Factors Influencing Transfection Efficiency, 써모 피셔 사이언티픽 코리아 주식회사,
https://www.thermofisher.com/kr/ko/home/references/gibco-cell-culture-basics/transfection-basics/factors-influencing-transfection-efficiency.html
Wikipedia. 2020. “Lipofectamine” 3 December. https://en.wikipedia.org/wiki/Lipofectamine.
Shaohui Cui, Yueying Wang, Yan Gong, Xiao Lin, Yinan Zhao, Defu Zhi, Quan Zhou, Shubiao Zhang, Correlation of the cytotoxic effects of cationic lipids with their headgroups, Toxicology Research, Volume 7, Issue 3, May 2018, Pages 473–479, https://doi.org/10.1039/c8tx00005k
Lipofectamine® Transfection Reagent, 써모 피셔 사이언티픽 코리아 주식회사, https://www.thermofisher.com/kr/ko/home/brands/product-brand/lipofectamine.html
Hyaluronan Reduces Cationic Liposome-Induced Toxicity and Enhances the Antitumor Effect of Targeted Gene Delivery in Mice, Yanping Qian, Xiao Liang, Jingyun Yang, Chengjian Zhao, Wen Nie, Li Liu, Tao Yi, Yu Jiang, Jia Geng, Xia Zhao, and Xiawei Wei
,ACS Applied Materials & Interfaces 2018 10 (38),32006-32016
RNA INTERFERENCE. Nobel Media AB 2021. Tue. 4 May 2021. https://www.nobelprize.org/prizes/medicine/2006/advanced-information/
Post-transcriptional gene silencing: conservation and sequences, LITERATURE FOCUS| VOLUME 5, ISSUE 12, P514, DECEMBER 01, 2000,
https://doi.org/10.1016/S1360-1385(00)01806-9
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