Molecular Biology. Practical Guide - Velikov V.A. 2013

Isolation of Total DNA from Cells
Isolation of DNA from Blood Leukocytes

Isolating total DNA from animal Cells is relatively straightforward because the Plasmalemma, nuclear, and mitochondrial membranes are "solubilized" in the presence of the anionic detergent sodium dodecyl sulfate (SDS), and animals lack the complex Cell wall found in plants, for example (see Practical 2.2). DNA can be released from the DNA-protein complex using proteinases or chaotropic salts.

Alternatively, phenol extraction of DNA can be performed, much like with Bacteria. Other substances will remain in solution during subsequent alcohol precipitation of DNA, making them easy to remove.

DNA can be isolated from any nucleated tissue, though the quantitative yield varies among tissue types. Blood is frequently used for DNA extraction. Unlike mature erythrocytes, Blood Leukocytes contain nuclei. Total DNA isolated from 100 µl of whole blood is sufficient for several restriction digests, PCR assays, and sequencing reactions. Mitochondrial DNA and RNA are also present in the resulting preparation. In the protocol described below, phenol is used to remove Proteins from the DNA. When molecular biologists use the term "phenol," they typically refer to a 1:1 mixture of Water-saturated phenol and chloroform rather than the crystalline substance. Chloroform enhances phenol extraction efficiency, while isoamyl alcohol suppresses foaming.

Materials and Equipment

Blood samples, microcentrifuge, freezer, incubator, micropipettes with tips, plastic microtubes.

Solutions

- SSC buffer. 3 M NaCl; 0.3 M sodium citrate, pH 7.0. To prepare 1 L of a 20× concentrated SSC stock: NaCl - 175.3 g; sodium citrate - 88.2 g. Adjusting the pH is generally unnecessary.

- 3 M sodium acetate, pH 5.2. For 40 ml: dissolve 9.85 g of sodium acetate in 20 ml of water, and adjust the pH to 5.2 with glacial acetic acid.

- 0.2 M sodium acetate, pH 5.2. Prepared by diluting the 3 M solution.

- 10% SDS solution. Dissolve 100 mg in 10 ml of distilled water.

- TE buffer (Practical 1.1).

- Phenol-chloroform mixture (Practical 1.1).

- Chloroform-isoamyl alcohol mixture (Practical 1.1).

Procedure

1. Add 2 volumes of distilled water to 100 µl of whole blood ^a to bring the final volume to 0.3 ml. Mix thoroughly and let stand for 15 minutes.

2. Centrifuge the sample for 10 minutes at 5000 rpm. Discard the supernatant.

3. Wash The Cell pellet with two volumes of SSC buffer by adding 200 µl of 1× SSC to the tube. Mix gently.

4. Centrifuge as in step 2. Discard the supernatant.

5. Add 54 µl of 0.2 M sodium acetate ^b and 6 µl of 10% SDS to the pellet. Thoroughly resuspend the cell pellet using a micropipette or vortex mixer. Incubate at 37°C for 0.5–1 hour to allow cell lysis.

6. Add 2 volumes of TE buffer and deproteinize the sample using phenol. To do this, add an equal volume of the phenol-chloroform mixture to the tube, shake well, and centrifuge. Transfer the aqueous phase (upper layer) to a clean tube without disturbing the interphase.

7. Repeat the procedure from step 6 using the chloroform-isoamyl alcohol mixture.

8. To the protein-free lysate, add 1/10 volume of 3 M sodium acetate, pH 5.2, mix, and precipitate the DNA by adding 2.5 volumes of cold 96% ethanol and placing the sample in a freezer at -20°C for 1–2 hours. Alternatively, the DNA can be left in alcohol overnight.

9. Centrifuge the sample for 10 minutes at maximum microcentrifuge speed. Discard the supernatant. Wash the pellet with 70% ethanol.

10. Air-dry the DNA pellet and dissolve it in 20 µl of TE buffer ^c.

Notes

^а Total DNA content in leukocytes ranges from 30 to 60 µg/ml of blood.

^б Salt should always be added to the sample prior to phenol deproteinization, as phenol extraction in a low-salt buffer can reduce the DNA yield due to sample losses.

^в Many Buffer solutions contain Tris-hydrochloride (Tris-HCl) because, unlike other buffer systems, Tris prevents Microbial growth in such buffers. A concentrated stock solution of acidic Tris-HCl salt at the desired pH is prepared by titrating Tris base (Tris-OH, tris(hydroxymethyl)aminomethane) with Hydrochloric acid.