Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
PROCEDURE:
1. Centrifuge cells in a microcentrifuge at approximately 1,500 X g for 2 min.
2. Aspirate the supernatant and resuspend the cells in 50 μl of TE Suspension.
3. Add 100 μl of NaOH/SDS, mix thoroughly (see Hint #1) or vortex for a couple of seconds.
4. Add 75 μl of Ammonium Acetate and vortex for a couple of seconds.
5. Incubate on ice for 10 min.
6. Centrifuge in a microcentrifuge at maximum speed for 3 min.
7. Save the supernatant and add 0.6 volumes of 100% Isopropanol
8. Incubate on ice for 10 min.
9. Centrifuge in a microcentrifuge at maximum speed for 10 min.
10. Aspirate supernatant without aspirating the DNA pellet.
11. Add 1 ml of 70% Ethanol, mix well and centrifuge in a microcentrifuge at maximum speed for 5 min.
12. Aspirate the supernatant without aspirating the DNA pellet.
13. Allow the DNA pellet to air dry for a couple of minutes.
14. Resuspend DNA in 100 μl of TE Buffer.
SOLUTION:
70% Ethanol
70% (v/v) Ethanol
7.5 M Ammonium Acetate
TE buffer
10 mM Tris
pH 8.0
1 mM EDTA
NaOH/SDS Solution
1% (w/v) SDS
0.2 M NaOH
TE Suspension
10 mM EDTA
25 mM Tris
pH 8.0
REAGENTS AND CHEMICALS:
SDS
Ammonium Acetate
Tris
Ethanol
EDTA
Isopropanol
Sodium Acetate