Biochemical experiment Gel extraction Ligation 4 th week

Biochemical experiment Gel extraction & Ligation 4 th week 1

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. 2

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+.

Method • Agarose Gel Extraction • 1. 정제할 DNA를 포함한 Gel 부분을 잘라낸다. • 2. 100 mg의 Gel Block을 1. 5 ml tube에 transfer -> 500 ul QGE buffer adding -> Incubation (60℃ , 51 min) Gel이 완전히 녹을 때까지 진행 -> 상온에서 cooling *가끔씩 섞어주면 Gel 이 더 잘 녹음 • 3. 2 ml Collection tube에 QGE column 장착 -> step 2 의 solution을 spin column에 첨가 -> cfg (13, 000 rpm, 30 sec) 내려간 Solution은 버리고 Spin column 다시 장착 • 4. Spin column에 W 1 buffer 400 ul 첨가 -> cfg (13, 000 rpm, 30 sec) 내려간 solution 제거 -> Spin column 다시 장착 -> spin column에 wash buffer 600 ul 첨가 후 1분 동안 incubation -> cfg (13, 000 rpm, 30 sec) 내려간 solution 제거 -> Spin column 다시 장착 • 5. cfg (13, 000 rpm, 3 min - 공회전) -> Collection tube 제거 Spin column을 새로운 1. 5 ml micro tube에 장착 • 6. TDW 30 ul 첨가 -> Incubation (Room Temperature, 2 min) -> cfg (13, 000 rpm, 2 min) -> Spin column 제거

Method • DNA Ligation • 1. Vector , Insert의 양을 정한다. (molar ratio of Insert(3) / Vector(1)) • 2. Vector(9 kb) Insert(0. 9 kb) 10 X reaction buffer T 4 Ligase TDW • 100 ng 30 ng 2 ul 1 ul Up to 20 ul 3. 16℃, overnight 으로 반응시킨다.