DNA Deoxyribo Nucleic Acid Phosphate Base Sugar Phospho

DNA (Deoxyribo Nucleic Acid) 구조 Phosphate Base Sugar Phospho diester bond (DNA가 음전하를 띄는 이유) Hydro bond (DNA의 염기쌍) DNA의 화학적 구조

전기영동 시 Gel의 선택 1) Gel의 종류에 따라 달라진다. - Agarose gel : 분자량이 100 bp~20 kbp, 해상력이 낮음 - Polyacrylamide gel(PAGE) : 분자량이 작을때(10 bp~1000 bp) 해상력이 높음 2) 핵산의 denaturation에 따라서 선택 - DNA : non-denaturating gel, 염기서열 분석시 denaturing gel 사용하기도 함 - RNA : Denautring gel (Alkaline, formaldehyde, Urea등 첨가) 3) 분리하고자 하는 물질에 따라 - DNA : Agarose gel, PAGE - RNA : Formaldehyde agarose gel - Protein : SDS-PAGE

Material • • • 전기영동장치 1 x TAE buffer 팁(tip) 마이크로피펫(micropipette) Ethidium bromide(Et. Br) UV Transilluminator 0. 8% agarose gel Loading buffer 증류수

Method Power H O 2 - ++ • Polymerized agarose is porous, allowing for the movement of DNA • Scanning Electron Micrograph of Agarose Gel (1× 1 µm)

Method DNA small - Power large +

Agarose Gel Buffer Flask for boiling Agarose

Electrophoresis Equipment Power supply Buffer Cover Gel tank Electrical leads Casting tray Gel combs Agarose

Agarose Gel Buffer Agarose Buffer Solution Agarose

Agarose Gel Pouring the gel Buffer Agarose

Agarose Gel Pouring the gel Buffer Agarose

Agarose Gel Pouring the gel Buffer DNA buffer wells Cathode (negative) Anode (positive) Agarose

Agarose Gel Pouring the gel Buffer Agarose

Agarose Gel Pouring the gel Buffer Agarose

Agarose Gel Pouring the gel Cathode (-) Buffer wells Bromophenol Blue DNA (-) Gel Anode (+) Agarose

DNA Ladder Standard 12, 000 bp 5, 000 DNA migration Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule bromophenol blue + 2, 000 1, 650 1, 000 850 650 500 400 300 200 100

Green Fluorescent Protein (GFP) u The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26. 9 k. Da) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. u Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. u In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. u In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. u. To date, the GFP many Bacteria, Yeast and gene has other Fungi, mammalian cells, including human. been fish introduced (such and as zebrafish), expressed plant, fly, in and

Subcloning Eco. RI GF P Kpn. I p. Litmus 28 GFP

Method n 1. subcloning할 plasmid DNA를 준비한다 n 2. plasmid DNA에 Enzyme을 처리한다 p. LT 28 -GFP 3 ul Eco. RI / Kpn. I 0. 5 ul / 0. 5 ul 10 X Reaction Buffer 2 ul TDW (up to 20 ul) 14 ul n 3. 37℃ 에서 10 ~ 20분 배양한다. n 4. 1% Agarose gel에 6 x DNA Loading Dye를 섞어 전기영동을 한다. ( Size 확인을 위해서 DNA Ladder를 같이 Loading 한다 ) Reaction Sample(2번 20 ul ) 6 X Loading Dye 4 ul n 5. Et. Br Staining 5 ~ 10분 n 6. 원하는 Size를 관찰한다. (UV lamp) n 7. Gel 을 잘라서 -20℃에 보관한다.