DNA Extraction (Using a commercial kit)
DNA or deoxyribonucleic acid is an organic chemical of complex molecular structure found in all
prokaryotic and eukaryotic cells as well as in many viruses. DNA carries the instruction needed to
create proteins which are essential in the development and functioning of the body as well as codes
genetic information for the transmission of inherited traits. Genomic DNA extraction is the process
of isolating DNA in a biological sample and is an important step in Polymerase Chain Reaction
(PCR).
There are 3 basic steps in DNA extraction i.e. (1) cell disruption by enzyme-detergent lysis, (2)
extraction with organic solvents and (3) DNA recovery by alcohol precipitation. Cell disruption uses
chemical reagents such as surfactants, lysis buffers and enzymes to solubilize lipids and proteins in
the cell membrane to create pores and lyse cells to release intracellular materials including DNA.
Detergents or surfactants such as sodium dodecyl sulphate (SDS), Triton-X and Tween series are
widely used in lysis buffers as they are capable of disrupting the hydrophobic-hydrophilic lipid
bilayer of the cell membrane. Detergents are often used along with enzyme proteinase K in lysing
animal tissue. Proteinase K is added to degrade DNA-associated proteins and other cellular proteins.
EDTA is also usually added as a chelating agent in order to reduce the activity DNase. EDTA
chelates the metals ions that serve as cofactor which increase the activity of nucleases. Addition of
organic solvents such as phenol/chloroform/isoamyl alcohol, facilitates separation of DNA from
other cellular components. Addition of alcohol (isopropanol and ethanol) followed by centrifugation
allows recovery and purification of DNA from the aqueous solution. The extracted DNA will then be
used as template for succeeding downstream applications such as polymerase chain reaction (PCR).
This procedure has been modified by biomedical companies to develop kits for efficient DNA
extractions. Here we present the process of isolating DNA from the fish tissue sample using the
Wizard® Genomic DNA Purification Kit by Promega Corporation.
The following materials and additional reagents are needed:
Micropipettor Sterile micropipette tips Sterile microcentrifuge tubes
Ice Water bath or incubator Centrifuge
Vortex 0.5M EDTA solution (pH 8.0) 20 mg/mL Proteinase K
Isopropanol 70% ethanol Spectrophotometer (or NanoDrop)
DNA Extraction Procedure
1. Mix 120µL 0.5M EDTA solution (pH 8.0) and 500 µL Nuclei lysis solution in a microcentrifuge
tube and chill on ice.
2. Sterilize dissecting tools by flaming. Excise 50 mg tissue from the specimen.
3. Place excised tissue in the chilled Nuclei lysis and EDTA solution
Add 17.5 µL of 20 mg/mL Proteinase K
4. Incubate at 55˚C for 3 hrs with vortex mixing one per hour. Make sure the tissue is completely
digested.
5. Add 3 µL RNase A solution and mix and incubate at 37˚C for 15 minutes. Cool to room
temperature.
6. Add 200 µL protein precipitation solution and vortex vigorously at high speed for 20 seconds.
Chill sample on ice for 5 minutes.
7. Centrifuge for 4 minutes at 13,000-16,000 x g.
8. Transfer the supernatant to a microcentrifuge tube containing 600 µL room temperature
isopropanol. Mix the solution by inversion until white thread-like strands of DNA form a visible
mass.
9. Centrifuge for 2 minute at 13,000-16,000 x g at room temperature. Discard supernatant.
10. Add 600 µL of room temperature 70% ethanol and gently invert several times to wash DNA
pellet.
11. Centrifuge at 13,000 x g for 2 minutes. Remove supernatant and allow drying for 10-15 minutes.
12. Add 100 µL of DNA rehydration solution and incubate at 65˚C for 1 hour.
13. Verify if extraction is successful by checking presence of genomic DNA using a
spectrophotometer, gel electrophoresis or NanoDrop.