Mr. Khadga Bikram Angbuhang
Faculty: Department of Microbiology
GoldenGate Intl College
Research
officer: LEAD Nepal.
Genomic
DNA extraction from animal tissues
Principle:
The isolation of DNA
usually begins with lysis of tissue or cells. Lysis is carried out in a salt
solution, containing detergents (SDS) and proteases (Proteinase K). SDS
solubilizes the cell membrane and lipid membranes of internal organelles and
denatures proteins (enzymes) which release chromosome in lysate. Treatment with
Proteinase K and Phenol: Chloroform can dissociate histones and other proteins
from DNA and precipitate them. Tris-HCL is a buffer, which retain constant pH;
ethylenediaminetetraacetic acid (EDTA), binds metal ions. Chloroform
solubilizes lipids and a lot of proteins and separate proteins and
polysaccharides from nucleic acids in the cell. Chloroform is denser than water
solutions and thus after spinning, Chloroform and water will separate into two
distinct phases. The lower phase will be Chloroform. This is the phase that
proteins and polysaccharides find most chemically attractive. The upper aqueous
phase will contain your DNA. Free DNA is recovered in aqueous solution after
centrifugation and concentrated by ethanol. DNA is less soluble in solutions
containing isopropanol than in solutions containing ethanol. Precipitation with
isopropanol is performed at room temperature to lessen the risk that solutes
like sucrose or sodium chloride will be co precipitated with the DNA. DNA does
NOT dissolve in ethanol (Chilled Ethanol). Cold alcohol is used to separate DNA
out of water-based solutions.
Material
Required:
1. Extraction
buffer: (NaCl 10mM; Tris-HCl 10mM; EDTA 10mM, pH 8.0)
2 2. SDS
- 10%.
- 3 M Sodium acetate, pH 4.8 by acetic acid
- Phenol: Chloroform (1:1)
- Chloroform: isoamyl alcohol (24:1)
- RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
- Proteinase K:
10 mg/ml stock
- 70% ethanol
- TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0;
Autoclave before use.
Procedure:
1.
Transfer about 10-20 mg animal
tissue to an MFT 1.5mL tube labeled with an identification number.
2.
Add liquid nitrogen and grind the
tissue for 1 minute with a pestle.
3.
Add 300μl Extraction buffer and 100μl
SDS 5%.
4.
Add 15μl Proteinase K.
5.
Grind the tissue with a pestel.
6.
Incubate the tube at 60°C for at
least 30 minutes.
7.
Add
1-2 µl of RNase and incubate for 10 min at RT.
8.
Add
equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10 min at RT.
9.
Centrifuge at 8,000 rpm for 15 min.
10. Carefully
decant the aqueous (top) phase to a clean microfuge tube.
11. Add
an equal volume of chloroform: isoamyl alcohol (24:1) and mix gently and Centrifuging at 8000 rpm for 5 min.
12. Decant
the aqueous phase to a clean microfuge tube and add 1/10 or equal volume of 3 M sodium acetate and
300μl 10
or equal volume of
isopropanol.
13. Mix gently inverting the MFT.
14. Leave in cold temperature
for 30 minutes.
15. Centrifuge at 13,000 rpm for 5 minutes.
16. Eliminate the supernatant and collect pellet (in same MFT).
17. Wash pellet adding
500μl ethanol 70%.
18. Centrifuge at 13,000 rpm for 5 minutes.
19. Eliminate the supernatant.
20. Dry the pellet for 10-15 minutes at 60°C to evaporate
remaining ethanol.
