Biochemical experiment Gel extraction & Ligation 4th week

Gel extraction In molecular biology, gel extraction or gel isolation is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. After extraction, fragments of interest can be mixed, precipitated, and enzymatically ligated together in several simple steps. This process, usually performed on plasmids, is the basis for rudimentary genetic engineering. After DNA samples are run on an agarose gel, extraction involves four basic steps: identifying the fragments of interest, isolating the corresponding bands, isolating the DNA from those bands, and removing the accompanying salts and stain.

DNA Ligation Ligation in molecular biology is the joining of two nucleic acid fragments through the action of an enzyme. It is an essential laboratory procedure in the molecular cloning of DNA whereby DNA fragments are joined together to create recombinant DNA molecules, such as when a foreign DNA fragment is inserted into a plasmid. The ends of DNA fragments are joined together by the formation of phosphodiester bonds between the 3'-hydroxyl of one DNA terminus with the 5'- phosphoryl of another. RNA may also be ligated similarly. A co-factor is generally involved in the reaction, and this is usually ATP or NAD+. Ligation in the laboratory is normally performed using T4 DNA ligase, however, procedures for ligation without the use of standard DNA ligase are also popular.

Method Agarose Gel Extraction 1. 정제할 DNA를 포함한 Gel 부분을 잘라낸다. 2. 100mg의 Gel Block을 1.5ml tube에 transfer -> 500ul QGE buffer adding -> Incubation (60℃ , 15 min) Gel이 완전히 녹을 때까지 진행 -> 상온에서 cooling *가끔씩 섞어주면 Gel 이 더 잘 녹음 3. 2ml Collection tube에 QGE column 장착 -> step2 의 solution을 spin column에 첨가 -> cfg (13,000rpm, 30sec) 내려간 Solution은 버리고 Spin column 다시 장착 4. Spin column에 W1 buffer 400ul 첨가 -> cfg (13,000rpm, 30sec) 내려간 solution 제거 -> Spin column 다시 장착 -> spin column에 wash buffer 600ul 첨가 후 1분 동안 incubation -> cfg (13,000rpm, 30sec) 내려간 solution 제거 -> Spin column 다시 장착 5. cfg (13,000rpm, 3min - 공회전) -> Collection tube 제거 Spin column을 새로운 1.5 ml micro tube에 장착 6. TDW 30ul 첨가 -> Incubation (Room Temperature, 2min) -> cfg (13,000rpm, 2min) -> Spin column 제거

Method DNA Ligation 1. Vector , Insert의 양을 정한다. (molar ratio of Insert(3) / Vector(1)) 2. 3. 16℃, overnight 으로 반응시킨다. Vector(9kb) 100ng Insert(0.9kb) 30ng 10X reaction buffer 2ul T4 Ligase 1ul TDW Up to 20ul