Preparation and concentration determination of plasmid DNA from

Preparation and concentration determination of plasmid DNA from E. coli : Mini-scale preparation 2014 -2학기 생화학실험(2) 담당교수 : 고재원 교수님 담당조교 : 정영진

Plasmid

Components of plasmid DNA Plasmid DNA component 1. Origin of replication 2. Selectable marker (Antibiotic Resistance gene) 3. Multiple cloning site ØP 53 (p. GEX-4 T-1) -> 394개 아미노산 = 1182 bp -> Total bp: 4969 + 1182 bp = 6151 bp

Plasmid DNA for gene cloning Principle of gene cloning 1. plasmid DNA replication 2. cell proliferation y co n lo 8~12 h, 37℃, 200 rpm Incubation

Plasmid DNA for gene cloning <Cell harvest> Types of preperation Mini Midi Maxi Mega Giga DNA yield(ug) 100 ug> 100 -350 500 -850 1500 -2500 7500 -10000 purification scale

Plasmid purification method Anion-Exchange Chromatography Method 각 Buffer의 역할 Buffer QBT (low salt): DNA binding 이때 용질과 고정상의 흡착을 일으키는 분자 사이의 인력은 수소결합 등이 있다. Buffer QC (medium salt): RNA, protein, carbohydrate등의 제거가능 Buffer QF (high salt): Salt가 DNA binding을 억제하여 elution이 가능

Materials Alkaline cell lysis P 1 Solution – Resuspension solution 25 m. M Tris-Cl p. H 8. 0 10 m. M EDTA, p. H 8. 0 100µg/ml RNase A P 2 Solution – Lysis solution 0. 2 M Na. OH 1%(W/V) SDS P 3 Solution – Neutralization solution 3 M Potassium acetate p. H 5. 5 Anion-exchange-based DNA purification Anion-exchange-based column Buffer QBT buffer-low salt Buffer QC buffer-medium salt Buffer QF buffer-high salt isopropanol 70% Et. OH D. W

Alkaline cell lysis method • Procedures 1) Resuspension the pellet in 0. 3 ml of Buffer P 1 2) Add 0. 3 ml of Buffer P 2 - Mix the contents thoroghly by inverting the tube several times(5 times). - Incubate the mixture at room temperature (18– 25 °C) for 5 min. 3) Add 0. 3 ml of Buffer P 3 - Mix the contents thoroghly by inverting the tube several times(5 times). - Incubate the mixture in ice for 5 min. 4) Centrifuge : 18000 x g, 4℃, 10 min 5) Column equilibration (Buffer QBT) 1 ml 6) Apply supernatant from step 4) 7) Wash column with Buffer QC 2 ml x 2 8) Elute DNA with Buffer QF 0. 8 ml 9) Precipitate DNA by adding 0. 56 ml(x 0. 7 volume of elution) RT isopropanol to eluted DNA and mix. centrifuge : 18000 x g, 4℃, 30 min. Carefully decant supernatant. 10) Wash DNA pellet with 1 ml RT 70% ethanol, centrifuge : 18000 x g, RT, 10 min. Carefully decant supernatant. 11) Air-dry pellet 10 min and redissolve DNA in 20µl D. W 12) Measure DNA concentration by Nano-drop.

Identification of purified plasmid DNA <Nano drop> ng/µl 의 단위로 확인 가능 Nano drop을 통해 plasmid DNA의 농도를 쉽게 구할 수 있다.

Identification of purified plasmid DNA Agarose gel loading 으로 purify 한 plasmid DNA의 state를 확인 할 수 있다. ex)- plasmid DNA의 size, impurity 여부 등