Isolation of Total DNA and RNA from Blood Using Phenol Chloroform Extraction

yujian623

Contributor:
Russell Ingersoll
University of California, Berkeley

Procedure:


1. Collect blood in Heparin containing vacutainer.

2. Dilute the collected whole blood 1:1 using 2X Extraction Buffer (final buffer concentration is 10 mM Tris, 1 mM EDTA and 1% SDS).

3. Mix to homogeneity by inverting the tube several times.

4. Add Proteinase K to a final concentration of 300 μg/ml and mix well.

5. Incubate 3 to 6 hr at 37°C.

6. Cool the solution to room temperature.

7. Add an equal volume of Phenol-TE, mix well by inversion, centrifuge at 4,300 X g for 15 min to separate the phases and save the upper (aqueous) phase.

8. To the aqueous phase, repeat the Phenol-TE extraction (Step #7).

9. To the aqueous phase add 1 volume of SEVAG, mix well by inversion, centrifuge at 4,300 X g for 15 min to separate the phases and save the upper (aqueous) phase.

10. To the aqueous phase add 0.1 volume 2 M NaCl (final concentration is approximately 0.2 M) and mix well by inversion.

11. Add 2 volumes of ice-cold 100% Ethanol and mix well by inversion.

12. Centrifuge at approximately 4,300 X g for 15 min at room temperature and decant the supernatant.

13. To the DNA/RNA pellet add 1 volume of 70% Ethanol and mix well by inversion.

14. Centrifuge at approximately 4,300 X g for 15 min at room temperature and decant the supernatant.

15. Remove the excess Ethanol with a Pasteur pipette allowing the Ethanol to slowly enter the pipette by capillary action.

16. Allow the DNA/RNA pellet to air dry for a few minutes.

17. Dissolve the DNA/RNA pellet in an appropriate volume of TE Buffer (usually around 100 μl per 1 ml of whole blood, see Hint #2).



Solutions


Proteinase K
1 mg/ml Proteinase K



Extraction Buffer (2X)
20 mM Tris, pH 7.4
2% (w/v) SDS
2 mM EDTA



TE Buffer
10 mM Tris, pH 7.4
1 mM EDTA



70% (w/v) Ethanol

2 M NaCl

SEVAG
24:1 Chloroform:Isoamyl Alcohol (CAUTION! see Hint #1)



Phenol-TE
OPTIONAL: 0.1% (w/v) 8-Hydroxyquinoline (CAUTION! see Hint #1)
TE Equilibrated Phenol (CAUTION! see Hint #1)





BioReagents and Chemicals:


Isoamyl Alcohol
Heparin
Chloroform
Proteinase K
8-Hydroxyquinoline
SDS
EDTA
Tris
Ethanol
Sodium Chloride


Protocol Hints:


1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The DNA/RNA pellet can be incubated at 37°C with gentle rocking to aid in the dissolving process.