Gel Purificatio

n

• also known as gel extraction or gel isolation

• is a technique used to isolate a desired fragment of intact DNA from an agarose gel

identifying the fragments of

interest

isolating the corresponding

bands

The 4 Basic Steps

The 4 Basic Steps

isolating the DNA from those bands

removing the accompanying salts and stain

The 4 Basic Steps

How It’s Done

The desired band is identified and

physically removed with a cover slip or

razor blade.

UV light is shone on the gel in order to illuminate all the

ethidium bromide-stained DNA.

How It’s Done

isolating and cleaning the DNA

fragment of interest

The removed slice of gel should contain the desired DNA

inside.

Isolating and Cleaning the DNA Fragment

Spin Column Extraction Dialysis Traditional

Spin Column Extraction

• the dissolution of the gel-slice

• application of the solution to a spin-

column • a 70% ethanol wash• elution of the DNA in a

small volume (30 µL) of water or buffer.

Dialysis

• The gel fragment is placed in a dialysis tube

that is permeable to fluids but impermeable to molecules at the size

of DNA.• An electric field is established around the

tubing • The tube solution can

then be pipetted out and will contain the desired

DNA with minimal background.

Traditional• placing the agarose fragment inside a folded pocket of Parafilm wax

paper• Physical compression,

partially liquifying the gel and its contents.

• liquid droplets can then be directed out of the

pocket • where they are pipetted

into a small tube• A butanol extraction

removes the ethidium bromide stain, followed by a phenol/chloroform

extraction of the cleaned DNA fragment.

Western Blot

• also known as protein immunoblot

• is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract.

Tissue preparation

Gel electrophoresis

Steps

Transfer Blocking

Steps

Detection Analysis

Steps

Steps

Uses

HIV Test or Lyme disease in blood

samples

Epitope mapping, amino acid

composition and sequence analysis

Prepared by:Jan del Rosario

_Super Yano