Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
PROCEDURE:
1. Purify at least 10 μg DNA by conventional Phenol extractions to remove any proteins from the DNA solution.
SOLUTION:
Acetate-Equilibrated Redistilled Phenol
3M Sodium Acetate
REAGENTS AND CHEMICALS:
CITATION AND /OR WEB RESOURCES:
2. Add 0.1 volume of 3 M Sodium Acetate and 2 volumes of Ethanol, and mix to precipitate the DNA.
3. Centrifuge in a microcentrifuge at full speed for 5 min to pellet the DNA.
4. Resuspend the DNA in 50 mM Sodium Acetate Buffer + 75 mM NaCl. The final volume should be 100 μl and the DNA concentration should be 100 μg/ml. To do this, resuspend the DNA in 9 parts water and add 0.5 parts 1 M Sodium Acetate, pH 4.0 and 0.5 parts 1.5 M NaCl.
5. Add an equal volume of Acetate-equilibrated Redistilled Phenol and mix by shaking for 2 to 3 min.
6. Centrifuge for 3 to 5 min in a microcentrifuge to separate the phases and recover the aqueous phase to a fresh tube (save the Phenol phase).
7. Repeat the Phenol addition to the aqueous phase, shake, and centrifuge two more times.
8. A considerable amount of supercoiled DNA can be recovered from the Phenol phase and interface by re-shaking the Phenol phase with an equal amount of 50 mM Sodium Acetate buffer + 75 mM NaCl.
9. Centrifuge the back-extracted Phenol phase for 3 to 5 min in a microcentrifuge to separate the phases and recover the aqueous phase. (It is optional to extract the aqueous phase with Chloroform to remove any traces of Phenol before Ethanol precipitating.) To perform a Chloroform extraction, add an equal volume of Chloroform:Isoamyl Alcohol, mix, and centrifuge for 3 to 5 min in a microcentrifuge to separate the phases.
10. Pool all of the aqueous phases and add 10 to 20 μg of carrier tRNA.
11. Add NaCl to 0.1 M and 2.5 volumes of Ethanol.
12. Mix well by inversion. There may be a large salt precipitate that forms during the Ethanol precipitation, which can be removed with low-speed centrifugation (1,000 rpm for 5 min in a microcentrifuge) and discarded. Centrifuge the sample at full speed for 5 to 10 min in a microcentrifuge to pellet the DNA.
13. Resuspend the DNA in TE buffer and adjust the NaCl concentration to 0.5 M.
14. Purify the DNA by chromatography over a G-50 column or equivalent.
Remove the acetate buffer and replace it with fresh acetate buffer. Shake several minutes. Collect redistilled Phenol directly into an equal volume 50 mM Sodium Acetate, pH 4.0 (add no NaCl to this solution). the shaking and buffer replacement 1 or 2 more times.
Repeat the shaking and buffer replacement 1 or 2 more times.
Chloroform : Isoamylalcohol (24:1)
TE Buffer - 10 mM Tris (pH 8.0), 1 mM EDTA
1.5 NaCl
1M Sodium Acetate Buffer - 82 g Sodium Acetate
Make up to 1 liter with H2O
Add Glacial Acetic Acid until the pH is about 4.0
EDTA
Isoamyl Alcohol
Chloroform
Tris
Phenol
Sodium Chloride
Ethanol
Glacial Acetic Acid
Sodium Acetate
Zasloff M, Ginder GD, Felsenfeld G. A new method for the purification and identification of covalently closed circular DNA molecules. Nucleic Acids Res 1978; 5:1139-52.