Preparation and concentration determination of plasmid DNA from

Preparation and concentration determination of plasmid DNA from E. coli : Midi-scale preparation 2013 -2학기 생화학실험(2) 담당교수 : 송재환 교수님 담당조교 : 이동주

Plasmid

Components of plasmid DNA Plasmid DNA component 1. Origin of replication 2. Selectable marker (Antibiotic Resistance gene) 3. Multiple cloning site ØP 53 (pc. DNA 3) -> 394개 아미노산 = 1182 bp -> Total bp: 5402 + 1182 bp = 6584 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℃, 210 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 EQU (low salt): DNA binding 이때 용질과 고정상의 흡착을 일으키는 분자 사이의 인력은 수소결합 등이 있다. Buffer WASH (medium salt): RNA, protein, carbohydrate등의 제거가능 Buffer ELU (high salt): Salt가 DNA binding을 억제하여 elution이 가능

Materials Alkaline cell lysis Solution I – Resuspension solution 50 m. M glucose 25 m. M Tris-Cl p. H 8. 0 10 m. M EDTA, p. H 8. 0 Solution II – Lysis solution 0. 2 N Na. OH 1%(W/V) SDS Solution III – Neuralization solution 5 M Potassium acetate Glacial acetic acid Anion-exchange-based DNA purification Anion-exchange-based column Buffer EQU buffer-low salt Buffer WASH buffer-medium salt Buffer ELU buffer-high salt isopropanol 70% Et-OH D. W

Alkaline cell lysis method • Procedures 1) Resuspension the pellet in 8 ml of Alkaline lysis solution I (Buffer RES) “ Vol. [ m. L ] = Culture Volume [ m. L ] x OD 600 / 50” - Culture volume: 100 ml , O. D 600: 1. 5 => 3 ml 2) Add 8 ml solution II (Buffer LYS) - 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) Column equilibration (Buffer EQU) 12 ml 4) Add 8 ml solution III (Buffer NEU) - inverting the tube several times(10 ~15 times) 5) Inverting the tube 3 times directly before applying the lysate to the equilibrate Nucleo. Bond® Xtra Column Filter 6) Wash column filter and column (Buffer EQU) 8 ml 7) Discard column filter

Anion-exchange chromatography method 7) Add 8 ml of Buffer WASH into column and allow the column to empty by gravity flow 8) Elution - Add 5 ml (Buffer ELU) 9) Add 3 ml isopropanol 10) Centrifugate for 30 min at 4500 rpm at 4˚C in a centrifuge 11) Carefully decant the supernatant. Transfer to microcentrifugal tube. 12) Add 1 ml of 70% Et-OH. 13) Centrifugate for 5 min at 13000 rpm, RT in a microcentrifuge 14) Carefully decant the 70% Et-OH. 15) Air dry 5 min and redissolve the DNA in 50~100 ul D. W.

Identification of purified plasmid DNA <Nano drop> ng/ul 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