unisel

UNIVERSITI SELANGOR

FACULTY OF SCIENCE AND
BIOTECHNOLOGY
(FasBIO)
MOLECULAR TECHNIQUES BBS 1231
Practical 3: Isolation of genomic DNA from animal
tissues
(LECTURER: MDM. Yasotha a/p Sundaraj)
GROUP 3
Devaraj a/l Ravindran 4111018161
Jeevitha a/p Tana sakaran 4111016861
Sujatha a/p kanniappan 4111009531
Premkumar a/l Subramaniam 4111017391
Theevindran a/l Kesavan 4111017311
Paveanthen a/l Ramachandran 4111023071
Mohamed Mustafa Osman 4102008031

INTRODUCTION:
Genomic DNA is the DNA that holds the complete set of genetic data for an organism.
Genomic DNA spans 46 chromosomes in human, there is a complete set of genetic
information including coding DNA and non-coding DNA. Coding DNA leads to expression
of genetic traits meanwhile non-coding DNA lacks this function. In Human Genome Project,
genomic DNA of humans has been sequenced to learn about the specific functions of various
areas of the genome. With the ability to locate specific genes and other information, it is
useful to diagnose and treat genetic conditions. The genomic DNA of some other organisms
has been sequenced too. By having the sequencing information, researchers can identify areas
in which genomic DNA differs from individual to individual. Many organisms have a
complete set of genomic DNA in every cell. In the cell, different operations determine which
part of the genome is active. These operations cause the organism to create differentiated
cells and regulate cell function. Sometimes, there can be a mistake in the regulation thus
causing malignancies and other genetic disorders.
AIM:
To isolate genomic DNA from animal tissue.
METHOD:
The DNA in the cell was released by breaking down the plasma membrane of the cell and
orgenelles by certain enzymatic reaction, then DNA was isolated via mechanical method
(centrifuging) and precipitated by using NaOAc to neutralize the charges on DNA . DNA
resuspended in TE buffer and stored at -20 degree Celsius for further usage.
MATERIALS:
Fresh tissue of chicken (muscle), Petri dish, 1.5 ml centrifuge tube, Lysis buffer, Phenol:
Chloroform: Isoamylalcohol (25: 24: 1), Chloroform: Isoamylalcohol (24:1), Proteinase K,
3M NaOAc (pH 6.0), absolute alcohol, 70% ethanol, TE buffer.
PROCEDURE:
1. About 500 mg of animal tissue was washed.
2. The tissue was minced by using scalpel on a Petri dish.
3. The minced tissue was transferred into a sterile 1.5 microcentrifuge tube and 500
micro litre of Lysis buffer was added.
4. 30 micro litre of Proteinase K was added and the suspension was incubated for 1 hour
at 55 degree Celcius in a water bath.
5. The digestion mixture was mixed with an equal volume of Phenol: Chloroform:
Isoamylalcohol (25: 24: 1) and centrifuged at maximum speed for 2 minutes.
6. An aqueous phase, a whitish interface and an organic solvent phase were observed.
The aqueous phase was transferred into fresh tube.
7. Equal volume of Chloroform: Isoamylalcohol (CIA; 24:1) was added and centrifuged
at maximum speed for 2 minutes.
8. The aqueous layer was transferred to another fresh tube.
9. 1/10 volume of 3M NaOAc (pH 6.0) was added followed by 1 volume of absolute
ethanol.
10. The solution was gently mixed and left it on ice to incubate for 30 minutes.
11. The DNA was pelleted by centrifuging at maximum speed for 30 seconds.
12. The pellet was washed with 1ml of 70% ethanol and air-dried at room temperature
for 15 minutes.
13. The pellet was resuspended in 40 micro litre of TE buffer and stored at -20 degree
Celsius for further use.

OBSERVATION:
1. The digestion mixture of the chicken tissue was centrifuged by adding equal volume
of PCI solution ( Phenol:Chloroform:Isoamylalcohol, 25:24:1 ). PCI solution was red
in colour and mixed with the digestion mixture. After being centrifuged, there were 3
layers of solution; aqueous phase, a whitish interface and organic solvent phase.




2. 460 micro litres of the aqueous phase solution obtained from the previous centrifuged
products and mixed with equal volume of CIA solution (Chloroform: isoamylalcohol,
24:1). CIA solution was milky. The centrifuged products of the mixture were double
layers of solutions whereby both of them were clear and considered to be supernatant
too.

Chicken digestion mixture Chicken tissue digestion mixture
after being centrifuged by
adding equal volume of PCI
solution

3. 800 micro litres of the super naked solution was obtained and mixed with 80 micro
litres of 3M NaOAc followed by 800 micro litres of absolute ethanol. The mixture is
then incubated on ice for at least 30 minutes and centrifuged at maximum speed or 30
seconds. After centrifuging, a pellet of DNA was obtained.

4. The pellet of DNA was washed with 70% Ethanol and mixed with TE buffer. Upon
mixing with TE buffer, the pellet of DNA dissolved in it and becomes concentrated in
liquid form.
DISCUSSION:
1. LYSIS BUFFER
Lysis buffer is used to break the cell wall and cell membrane of the cell to analyze the
compounds of the cells. There are many kinds of lysis buffer for different purposes
such as red blood cell lysing and DNA extraction.

2. PROTEINASE K
Proteinase K has the ability to digest protein and also deactivate enzymes such as
DNase and RNase . It is widely used to digest protein and remove contamination during DNA
extraction. By increasing the reacting temperature the enzymatic also increases. Proteinase
K is also stable over wide pH range (4-12) and optimum of pH (7.5-12).

3. PCI, Phenol: Chloroform: Isoamylalcohol (25:24:1)
PCI solution is used to extract DNA via phase separation, it contains Phenol which is
water soluble and gives an unclear interface, with the help of chloroform it can be
Centrifuged products of the mixture
of equal volume of CIA solution and
the aqueous phase solution
DNA pellet
sharpened. Meanwhile Isoamylalcohol reduces foaming. This solution also contains
antioxidants because oxidised phenol can damage nucleic acids. Isoamylalcohol also
functions to stabilize chloroform, this is because exposure of pure chloroform to
oxygen and UV light produce phosgene gas.

4. CIA, Chloroform: Isoamylalcohol (24:1)
CIA is a type of detergent that discards proteins and lipids in the cell membrane by
dissolving it. As the proteins and lipids are dissolved, the bond within the cell
membrane is disrupted thus causing the breakdown of cell membrane. CIA will then
form complexes with them and precipitated out from the solution.

5. SODIUM ACETATE, NaOAc
Sodium Acetate is a kind of salt, it helps to precipitate out DNA by neutralising the
charges on the sugar phosphate bone of DNA thus making it less hydrophilic and
therefore less soluble in water.

6. ABSOLUTE ETHANOL
Absolute Ethanol also helps in precipitating DNA, it helps sodium acetate to interact
more easily with the DNA. Then, DNA gets less hydrophilic and eventually less
soluble in water and precipitated.

7. TE BUFFER
TE buffer prevents the DNA from degradation, thus DNA can be stored in
concentrated form for later usage.

CONCLUSION:
The genomic DNA from the chicken tissue was successfully isolated after going through the
procedure carefully.

REFERENCE:
1. Title: Lysis buffer, Accessed from: http://en.wikipedia.org/wiki/Lysis_buffer,
Accessed date: 10/9/2011.
2. Title: Proteinase K, Accessed from: http://en.wikipedia.org/wiki/Proteinase_K,
Accessed date: 10/9/2011
3. Title :PCI solution, Accessed from:
http://en.wikipedia.org/wiki/Phenol%E2%80%93chloroform_extraction, Accessed
date: 10/9/2011
4. Title: CIA solution, Accessed from:
http://wiki.answers.com/Q/What_is_the_function_of_chloroform_isoamyl_alcohol,
Accessed date: 10/9/2011
5. Title: Sodium Acetate salt, Accessed from: http://bitesizebio.com/articles/the-basics-how-
ethanol-precipitation-of-dna-and-rna-works/, Accessed date: 10/9/2011
6. Title: Ethanol, Accessed from: http://bitesizebio.com/articles/the-basics-how-ethanol-
precipitation-of-dna-and-rna-works/, Accessed date: 10/9/2011
7. Title: TE buffer, Accessed from:
http://wiki.answers.com/Q/What_is_the_role_of_TE_buffer_in_DNA_isolation,
Accessed date: 10/9/2011.