Phenol Chloroform Extraction


1. Turn on heat block to 55°C.
2. Add 500 μl of STE buffer – the same tip can be used for all tubes.
3. Pick out one piece of muscle (~2x2 mm), liver or a fin clip. Use an open flame to clean the cutting equipment after each sample is taken.
4. Add 15 μl of 1% proteinase K to each tube. Proteinase K is heat stable, and therefore it is not necessary to keep on ice. Use a new tip for each tube.
5. Add 75 μl of 10% SDS to each tube.
6. Thoroughly mix contents of the tubes by inversion.
7. Place samples in the heat block (55°C) till the tissue is completely dissolved (up to overnight or add more proteinase K). Mix samples by inversion periodically to dislodge any solid pieces.
8. Add 600 μl of equilibrated phenol (pH > 8.0).
9. Mix by inversion for 2 minutes.
10. Centrifuge samples at full speed (14,000 rpm) for 10 minutes.
11. Collect aqueous phase into the newly labeled tubes – be conservative by leaving what can not be cleanly collected.
12. Add 600 μl of equilibrated phenol (pH > 8.0).
13. Mix by inversion for 2 minutes.
14. Centrifuge samples at full speed (14,000 rpm) for 10 minutes.
15. Collect aqueous phase into the newly labeled tubes – be conservative by leaving what can not be cleanly collected.
16. Add 600 μl of chloroform:isoamyl alcohol (24:1) mix.
17. Mix by inversion for 2 minutes.
18. Centrifuge samples at full speed (14,000 rpm) for 10 minutes.
19. Collect aqueous phase into the newly labeled tubes – be conservative by leaving what can not be cleanly collected.
20. After this step, absolutely no chloroform should remain the tubes.
21. Add 55 μl of 5M NaCl to each tube (10% total volume).
22. Add 1000 μl of absolute (or 95%) alcohol to the aqueous phase.
23. Thoroughly mix by inversion.
24. Precipitate the samples overnight (at least 1 hour) in -20°C freezer.
25. Centrifuge for 10 minutes at full speed (14,000 rpm).
26. Pour off alcohol, leaving the pellet of DNA behind.
27. Wash the pellet by adding ~500 μl of 70% alcohol. The same tip can be used if the alcohol is dribbled in from above the tube.
28. If the DNA pellet becomes loose, spin for 2 minutes at 14,000 rpm.
29. Remove all alcohol, leaving the pellet of DNA behind.
30. Let the pellets either air dry or dry in the speedwac.
31. When the tubes are dry resuspend the pellet in 50 μl of water and let dissolve.
32. Store suspended DNA in -20°C freezer.

 

Notes


1. Phenol is a dangerous substance that will burn you if it gets on your skin. WEAR GLOVES and BE CAREFUL. Work in a hood if possible.
2. Equilibrated phenol is best purchased from commercial sources. The pH is important since chromosomal DNA will end up in the phenol phase if the pH is acid (around pH 5).
3. Be careful to determine which layer is the phenol. The density of pure phenol is almost identical to H20. Small changes in the density of your water layer (excess salt, e.g.) can lead to layer inversion.

 

Extraction examples

DNA

DNA extraction with phenol/chloroform/isoamyl alcohol pH 8 - aqueous top phase contains the majority of DNA, interphase mostly proteins, and lower organic phase most of the RNA and lipids

DNA

Left: phenol/chloroform extraction of DNA from mouse tail sample; some remnants of hair from proteinase K digestion of tail clip. Right: parallel extraction from mouse liver sample; characteristic red color of liver tissue partly from hemoglobin and other heme- and iron-containing proteins. Additional phenol extraction is recommended.

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