This presentation is about Gel extraction and Western blot.

1. Gel
Purification

3. The 4 Basic Steps
identifying the
fragments of
interest
isolating the
corresponding
bands

4. The 4 Basic Steps

5. The 4 Basic Steps
isolating the DNA
from those bands
removing the
accompanying
salts and stain

7. 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.

8. How It’s Done
isolating and
cleaning the DNA
fragment of interest
The removed slice of
gel should contain
the desired DNA
inside.

9. Isolating and Cleaning
the DNA Fragment
Spin Column
Extraction
Dialysis Traditional

10. 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.

11. Western
Blot

14. Tissue
preparation
Gel
electrophoresis
Steps

15. Steps
Transfer Blocking

16. Steps
Detection Analysis

17. Steps

18. Uses
HIV Test or Lyme
disease in blood
samples
Epitope mapping,
amino acid
composition and
sequence analysis

19. Prepared by:
Jan del Rosario
_Super Yano