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This presentation is about Gel extraction and Western blot.
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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