DNA Extraction
Content
Materials: Lysis solution: 1M NaCl 0.5M Na2 EDTA 1M Tris 9.3504g 0.2960g 0.8456g
Make up to 400 mL using double distilled water, autoclaved and stored at room temperature. 10% SDS 2.5g of SDS was dissolved in autoclaved double distilled water and made up to 25 mL and stored at room temperature. 6M NaCl (Mol. Wt – 58.44): 35.064g of NaCl dissolved and made up to 100mL with distilled water. The solution was autoclaved at 15 psi for 15 minutes and stored at room temperature. Ethyl alcohol (absolute): This was commercially purchased from Tedia and stored in the refrigerator. 70% ethanol: Absolute ethyl alcohol Autoclaved double distilled water Tris-EDTA (TE) buffer (pH-8.2) Tris 0.12114g Na2 EDTA 0.037224g Both were dissolved in 100 mL of the double distilled water, autoclaves and stored. Methodology: The cells were transferred to a sterile 15 mL centrifuge tube. The cells were centrifuged to pellet out. Then 3 mL of lysis solution was added to the pellet and aspirated. 200 µL of 10% Sodium Dodecyl Sulphate was added and the tube was gently inverted for 10 minutes. The tube was incubated for 16 ± 2 hours (overnight) in a 37° C water bath. 1mL of 6M NaCl was added to the tubes and inverted several times for 20 seconds. The tubes were centrifuged at 3,000 rpm for 20 minutes. The supernanant was carefully transferred to a new sterile 15 mL centrifuge tube. Double volume of ice-cold absolute ethanol was added to each tube and the tubes were inverted several times until the DNA precipitated. 7 0 mL 30mL
The precipitated DNA samples were transferred to 1.5 mL microfuge tube and centrifuged at 10,000 rpm for 5 minutes the supernatant was decanted and 1 mL of 70% ethanol was added and inverted for couple of times. The tube was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded without leaving any trace of the liquid. The pellet was air dried at room temperature. 100 µL of TE buffer was added to the semidried DNA pellet and left at room temperature overnight to dissolve. The samples were stored at 4° C.
Make up to 400 mL using double distilled water, autoclaved and stored at room temperature. 10% SDS 2.5g of SDS was dissolved in autoclaved double distilled water and made up to 25 mL and stored at room temperature. 6M NaCl (Mol. Wt – 58.44): 35.064g of NaCl dissolved and made up to 100mL with distilled water. The solution was autoclaved at 15 psi for 15 minutes and stored at room temperature. Ethyl alcohol (absolute): This was commercially purchased from Tedia and stored in the refrigerator. 70% ethanol: Absolute ethyl alcohol Autoclaved double distilled water Tris-EDTA (TE) buffer (pH-8.2) Tris 0.12114g Na2 EDTA 0.037224g Both were dissolved in 100 mL of the double distilled water, autoclaves and stored. Methodology: The cells were transferred to a sterile 15 mL centrifuge tube. The cells were centrifuged to pellet out. Then 3 mL of lysis solution was added to the pellet and aspirated. 200 µL of 10% Sodium Dodecyl Sulphate was added and the tube was gently inverted for 10 minutes. The tube was incubated for 16 ± 2 hours (overnight) in a 37° C water bath. 1mL of 6M NaCl was added to the tubes and inverted several times for 20 seconds. The tubes were centrifuged at 3,000 rpm for 20 minutes. The supernanant was carefully transferred to a new sterile 15 mL centrifuge tube. Double volume of ice-cold absolute ethanol was added to each tube and the tubes were inverted several times until the DNA precipitated. 7 0 mL 30mL
The precipitated DNA samples were transferred to 1.5 mL microfuge tube and centrifuged at 10,000 rpm for 5 minutes the supernatant was decanted and 1 mL of 70% ethanol was added and inverted for couple of times. The tube was centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded without leaving any trace of the liquid. The pellet was air dried at room temperature. 100 µL of TE buffer was added to the semidried DNA pellet and left at room temperature overnight to dissolve. The samples were stored at 4° C.
