DNA EXTRACTION

from ANIMAL
TISSUES
Sahara Nesli Gonzaga
Bernadette Diane Vista
Group 5, WAD1
Introduction:
• DNA, or deoxyribonucleic acid, is
the hereditary material in humans
and almost all other organisms.

 the continuing storage of

information: comprises
instructions needed for the
synthesis of other cell organelles
and other constituents.
Introduction:

 composed of two long

polymers of simple units, the
nucleotides, with sugar and
phosphate groups linked
together by ester bonds as the
backbone
Introduction:
• Extraction of DNA: removal of DNA
from cells or viruses from which it
normally inhabits.

• Norman Wang Method: one step

extraction of DNA from agarose gel
electrophoresis without Gel Cutting
& Purification.
Introduction:
• UVSpectrophotometry:
quantitative measurement of the
reflection or transmission properties
of a material as a function of
wavelength.

• Agarose Gel Electrophoresis:

separate DNA fragments based on
its molecular weight, charge and
shape.
Methodology:
Extraction of DNA
0.5mL aliquots + 0.5mL
8mL Blood + EDTA-Na
Wang’s lysis solution

Pellet + 1mL lysis Centrifuge (10,000 x g;

solution 20s)

Mix & centrifuge Pellet and Supernatant

(10,000 x g; 20s) (repeat procedure)
Methodology:
Extraction of DNA
Pellet + 0.2mL enzyme
Incubated (37o ; 10min)
solution

+0.3ml sodium iodine + 10μl Proteinase K

(NaI) solution solution ; incubated

+ 0.5ml isopropanol Mixed by inversion

Methodology:
Extraction of DNA
Centrifuge (10,000 x g; remaining alcohol was
20s) aspirated

Centrifuge (10,000 x g; Pellet + 1ml of

5min) isopropanol

Repeat procedure: Pellet + 0.1ml TE (Tris-

70% ethanol EDTA)
 Methodology:
UV spectrophotometer

5uL of DNA sample

diluted in 495 uL of TE
buffer

Recording of
absorption readings
http://www.spectralabsci.com/images/PE-UV-Vis-Spectrophotometers-Lambda
-12.jpg
 Methodology:
Agarose Gel Electrophoresis
Powdered agarose: 1x
TAE

+ EtBr (0.5ug/ml)

comb was placed (30-

http://upload.wikimedia.org/wikipedia/commons/a/a6/Gel_electrophoresis_app
aratus.JPG
40min)
 Methodology:
Agarose Gel Electrophoresis
+1x TAE buffer (depth of
at least 1mm)

sample DNA mixed with

the desired gel loading
buffer

viewed under UV light with

the use of a UV
http://upload.wikimedia.org/wikipedia/commons/a/a6/Gel_electrophoresis_app
aratus.JPG transilluminator.
Results & Discussion
Results & Discussion
Results & Discussion
Results & Discussion
Conclusion:
• DNA is an important type of nucleic
acid essential to any living organisms
metabolism, function and
development.

• There are numerous ways to extract

DNA depending upon the sample
from which it will be taken.
Conclusion:
• UV spectrophometer basically aids
in analyzing DNA extract through its
absorbance (ability to absorb light) in
a given wavelength

• Agarose Gel Electrophoresis

separates molecules through their
difference in charge, size and shape.
Guide Questions:
Guide Questions:
2. What is the principle behind agarose gel
electrophoresis?

 separates molecules based upon charge, size and

shape
 A direct current power supply is connected to the
electrophoresis apparatus and current is applied.
 The higher the applied voltage, the faster the
samples migrate.
 The buffer serves as a conductor of electricity and to
control pH.
Guide Questions:
2. What is the principle behind agarose gel
electrophoresis?

 Agarose is a polysaccharide derivative of agar:

mixture of agarsoe and hydrocoloids.
 Molecules having a more compact shape can move
more easily through the pores.
 Charge, size, shape together with buffer conditions,
gel concentration and voltage, affects the mobility of
molecules in gels.
Guide Questions:
3. Suggest other ways to purify animal DNA aside from
the above-mentioned procedure and commercially
available kits.

 Purification by silica: DNA of interest can be isolated

by virtue of its ability to bind silica in the presence of
high concentrations of chaotropic salts.
 Plasmid DNA purification: the difference in
denaturation and renaturation characteristics of
covalently closed circular plasmid DNA and
chromosomal DNA fragments.
Guide Questions:
3. Suggest other ways to purify animal DNA aside from
the above-mentioned procedure and commercially
available kits.

 Genomic DNA isolation: solution-based

Wizard® Genomic DNA Purification Kit, relies on a
series of precipitation steps to purify high-molecular-
weight DNA from a prepared lysate.
References:
• Carpi, A., & Ph.D.. (n.d.). Nucleic Acids. Visionlearning.
Retrieved February 15, 2011, from
http://www.visionlearning.com/library/module_viewer.php
?mid=63
• DNA Purification Protocols and Applications Guide.
(n.d.). Promega Corporation - Precision
• DNA structure. (n.d.). Wickipedia. Retrieved February
15, 2011, from
commons.wikimedia.org/wiki/File:DNA_Structure.jpg
http://ghr.nlm.nih.gov/handbook/basics/dna
References:
• Principle and practice of agarose gel electrophoresis.
(n.d.). gannon. Retrieved February 15, 2011, from
www.gannon.edu/resource/dept/sim/new/biologyexp_file
s/bio_exp_pdf/electrophoresis%20-%20principles
%20and%20practi
• Design for Life. Retrieved February 15, 2011, from
http://www.promega.com/paguide/chap9.htm
• Principles of Gel Electrophoresis. (n.d.).
arbl.cvmbs.colostate.edu. Retrieved February 15, 2011,
from
http://www.vivo.colostate.edu/hbooks/genetics/biotech/g
els/principles.html