DNA Extraction from Plasma and Serum

1. Introduction

There are occasions where the only materiel available on a patient is stored plasma or

serum samples. In normal individuals, the amount of DNA in these samples is very low

but sufficient to serve as template for PCRs. Moreover, increased amounts of circulating

DNA have been found in a variety of disorders, including cancer, autoimmune disease,

and infection. Additionally, small amounts of fetal DNA have been detected in maternal

plasma/serum during gestation. We have used the following protocol to successfully

genotype archival plasma samples.

2. Materials

1. 10X SDS /Protein K: (Lauryl sulphate [SDS] 10 g/100 mL, Proteinase K 5 mg/mL).

2. TE (Tris EDTA) buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

3. Phenol��chloroform (1��1 v/v).

4. Glycogen (10 mg/mL).

5. 7.5 M Ammonium acetate.

6. 100% ethanol.

7. 70% ethanol.

3. Method

1. Place 1.5 mL of serum or plasma into a 15-mL centrifuge tube.

2. Add 1.5 mL of 1X SDS proteinase K solution in the tube containing the serum and mix

well.

3. Digest overnight at 55°C in water bath.

4. Add 3 mL of phenol/chloroform solution.

5. Vortex 30 s and centrifuge for 10 min at 1000g using a swing-out rotor.

6. Transfer aqueous layer to fresh tube and repeat steps 4 and 5.

7. Transfer aqueous layer to fresh tube and add 5 ìL of glycogen (10 mg/L), 1 mL of 7.5 M

ammonium acetate, and 8 mL of 100% ethanol.

8. Mix by inverting and centrifuge at 2500g for 40 min.

9. Carefully remove supernatant and wash pellet in 10 mL of 70% ethanol.

10. Centrifuge at 2500g for 10 min. Carefully remove last traces of ethanol, and allow to air

dry for 10 min before redissolving in 100 ìL of TE.