Mammalian DNA prepared according to this protocol is approximately 20-50 kb and is suitable for use as a template for PCR reactions. the DNA yield varies between 0.5 and 3.0 μg/mg of tissue or 5-15 μg/300 μl of whole blood. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Rapid isolation of mammalian DNA
Principle
Mammalian DNA prepared according to this protocol is approximately 20-50 kb and is suitable for use as a template for PCR reactions.The DNA yield varies between 0.5 and 3.0 μg/mg tissue or 5-15 μg/300 μl whole blood.
Materials and Instruments
RNase without DNase Proteinase K Mammalian tissue or whole blood Move I. Materials For more product details, please visit Aladdin Scientific website.
Cell lysis buffer Ethanol Isopropanol Potassium acetate solution Erythrocyte lysis buffer TE
Suction device with vacuum tube Microcentrifuge wand Water bath
1. Buffers and solutions
Cell lysis buffer (10 mmol/L Tris-Cl (pH 8.0), 1 mmol/L EDTA ( pH 8.0), 0.1% (m/V) SDS)
Ethanol
Isopropyl alcohol
Potassium acetate solution (60 ml of 5 mol/L potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml water)
Erythrocyte lysis buffer (20 mmol/L Tris-Cl (pH 7.6))
TE (pH 7.6)
2. Enzyme and buffer
RNase without DNase (4 mg/ml)
Protease K (20 mg/ml)
3. Specialized equipment
Suction device with vacuum tube
Microcentrifuge rods
Water bath pre-set to 55°C or 65°C
4. Cells and tissues
Mammalian tissues or whole blood
II. Methods
1. Prepare tissue or whole blood for DNA isolation.
(1) Tissue
(1) Tissue ① Cut off 10-20 mg of tissue.
② Chop the tissue with a razor or scalpel or freeze the tissue in liquid nitrogen and grind it into powder with a mortar pre-cooled in liquid nitrogen.
(2) Blood
① Transfer 300 μl of whole blood sample to each of two microcentrifuge tubes. Add 900 μl erythrocyte lysis buffer to each tube, cover and mix upside down. Leave the solution at room temperature for 10 min, mixing several times.
② Centrifuge at maximum speed for 20 s at room temperature.
③ Keep 20 μl of supernatant in each tube and discard the rest.
④ Resuspend the leukocyte precipitate with the remaining small amount of supernatant. Combine the precipitates from the two tubes into one tube.
2. Transfer the chopped tissue or resuspended leukocyte precipitate to a centrifuge tube with 600 μl of ice-cold Cell Lysis Buffer. Mix the suspension by rapidly grinding it 30-50 times with a microprobe.
3. (Optional) Add 3 μl of Proteinase K to the lysate to increase the yield of genomic DNA. Allow the digest to stand at 55℃ for more than 3 h, but not more than 16 h.
4. Bring the digest to room temperature and add 3 μl of 4 mg/ml DNase-free RNase. 37°C for 15-60 min.
5. Bring the sample to room temperature. Add 200 μl of potassium acetate solution and shake vigorously for 20s to mix thoroughly.
6. Centrifuge the sample at maximum speed at 4°C for 3 min to precipitate the protein/SDS complex.
7. Transfer the supernatant to a new centrifuge tube with 600 μl isopropanol. Mix well and centrifuge at maximum speed for 1 min at room temperature to collect the DNA precipitate.
8. Pipette off the supernatant and add 600 μl of 70% ethanol. Invert the tube several times and centrifuge at maximum speed for 1 min at room temperature.
9. Carefully aspirate the supernatant and dry the DNA precipitate in air for 15 min.
10. Dissolve the DNA precipitate in 100 μl TE (pH 7.6).