DNA EXTRACTIONBy

PHENOL-CHOLOROFORM METHOD

A phenol-chloroform extraction is a liquid-liquid extraction. A liquid-liquid extraction is amethod that separates mixtures of molecules based on the differential solubilities of theindividual molecules in two different immiscible liquids. Liquid-liquid extractions arewidely used to isolate RNA, DNA, or proteins

Introduction

DNA extraction is done for:1.Tissue typing for organ transplant.2.Detection of pathogens3.Human identity testing4.Genetic research5.PCR6.Restriction fragment length polymerization.7.Hyberdization method.

APPLICATIONS

Cell cycle has four phases 1. Lag phase 2. log phase 3. Stationary phase 4. Death phase

State of DNA

Primary metabolites are produced during log phase and secondary metabolites are produced during stationary phase.

Cells for DNA extraction are extracted from log phase and early stationary phase.

METABOLITES

Step1: For DNA extraction 1.5ml culture is taken in appendroff tubes. It is centrifuged at 10,000 rpm for 3 minutes until pellets are formed. Step 2:

Supernatant is discarded Step3:

Cell pellets are washed with 300 µl 8.1 TE buffer. Mix well by vortex.

PROCEDURE

TE buffer is composed of 1. TRISMA 2. EDTA

TRISMA : maintains the pH of TE buffer at 8.1

EDTA: chelates Mg++ ions due to which cell membrane structure is distorted.

Composition of TE buffer

Step4: After washing with TE buffer, 30 µl SDS solution and 3 µl proteinase- k is added. After adding both of them mix them thoroughly by doing vortex. Incubate it at 37ºC for 1hour In water bath. A. Proteinase-K: Proteinase-K is used to degrade proteins.

• SDS and Proteinase-K

B. SDS: Sodium dodecyl sulfate. 1. Is anionic solution. 2. It causes cell lysis. Attack membrane lipids. 3. It also denatures proteins and lipids.

SDS

Step5: Now add 100µl 5M Nacl and 80µl CTAB. Mix thoroughly and incubate tubes at 65ººC for 10 minutes. A. Nacl: mask phosphate to bring DNA close together to form aggregates.

• Nacl and CTAB

B. CTAB: CETILE TRIMETHYL AMONIUM BENZOATE 1. Binds with DNA and form insoluble bonds. 2. Denatures lipid and proteins of cell.

• CTAB

Step 6:Add 700-800µl chloroform isoamyl alcohol and centrifuge it for 15 minutes. Chloroform: Binds with lipids and protein to precipitate them.Isoamyl: Anti foaming agent.

CHLOROFORM ISOAMYL ALCOHOL

After centrifugation two phases are formed. 1.Upper layer: Upper layer is called aqueous layer that contains DNA, RNA and Plasmid. 2. Lower layer: Is organic layer which contains proteins and lipids

Two phases

Step 7: After centrifugation aqueous phase is transferred to another appendroff tube. To this tube add 700-800 µl phenol chloroform isoamyl alcohol. Centrifuge again for 15 minutes.

PHENOL CHOLOROFORM ISOAMYL ALCOHOL

PHENOL: Precipitates protein and lipids CHLOROFORM:

Chloroform binds to non aqueous compounds i.e. lipids and proteins and precipitate them. ISOAMYL:

Its an anti-foaming agent.

Step 8: After centrifugation again transfer aqueous layer into another eppendorf tube. Add 600 µl isopropanol to it and again centrifuge for 15 minutes. ISOPROPANOL: Dehydrates DNA and forms weak DNA pellets.

ISOPROPANOLE

Step 9: After centrifugation remove supernatant and wash pallets with 70% ethanol. ETHANOL: Pellets produced by ethanol are stronger but less number of pellets are produced.

ETHANOL

Step 10: Supernatant is removed and pallets are air dried. Add 100 µl TE buffer . Aqueous phase contains isolated DNA. Use isolated strains for subsequent experiment or store at 20ºC.

TE BUFFER

Always add Proteinase-K before adding SDS because SDS causes bacterial culture to become viscous. After addition of SDS solution becomes clear.

Many DNA isolation methods don’t require vortexing because of shearing of DNA but in vortexing for 2 to 3 minutes can cause protein denaturing.

IMPORTANT POINTS