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.
Chicken digestion mixture
Chicken tissue digestion mixture after being centrifuged by adding equal volume of PCI solution
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.
Centrifuged products of the mixture of equal volume of CIA solution and the aqueous phase 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.
DNA pellet
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
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-howethanol-precipitation-of-dna-and-rna-works/, Accessed date: 10/9/2011 6. Title: Ethanol, Accessed from: http://bitesizebio.com/articles/the-basics-how-ethanolprecipitation-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.
