1.  LYSIS:   1 ml DNAzol BD + 0.5 ml of whole blood.

2.  DNA PRECIPITATION:   lysate + 0.4 ml isopropanol at 6,000 g x 6 min.

3.  DNA WASH:   0.5 ml DNAzol BD at 6,000 g x 5 min.;       1 ml 75% ethanol at 6,000 g x 5 min.

4.  DNA SOLUBILIZATION:   8 mM NaOH or water.

1.  LYSIS
Mix 1.0 ml of DNAzol BD with 0.5 ml of whole blood. Shake vigorously by hand for 15-20 sec and store at room temperature for 5 minutes. Stored blood samples have to be mixed well before taking an aliquot for the DNA isolation.

2.  DNA PRECIPITATION
Add 0.4 ml of isopropanol to the DNAzol BD-blood lysate. Vortex or shake vigorously and store for 5 minutes at room temperature. Sediment the precipitated DNA by centrifugation at 6,000 g for 6 minutes.

The volume of isopropanol used for the precipitation equals 0.4 volume of DNAzol BD used for the lysis. Vigorous mixing of the DNAzol BD-blood lysate with isopropanol dissolves protein aggregates and improves quality of the isolated DNA.

3.  DNA WASH
After centrifugation, remove the supernatant and add 0.5 ml of DNAzol BD to the DNA pellet. Vortex or shake the DNA pellet until it is completely dispersed. Centrifuge the resulting mixture at 6,000 g for 5 minutes. Remove the supernatant and wash the DNA pellet by mixing with 1 ml of 75% ethanol and centrifuge at 6,000 g for 5 minutes.

When using microcentrifuge tubes with snap caps, use a cotton swab to remove any residual blood accumulated in the cap and around the top of the tubes.

4.  DNA SOLUBILIZATION
Decant the ethanol wash and store the tubes vertically. Remove any residual ethanol with a micropipete. Do not allow the pellet to dry. Dissolve the DNA pellet in 200 µl of 8 mM NaOH and incubate at room temperature for 3 - 5 minutes followed by repetitive pipetting or vortexing. Neutralize the alkaline DNA solution with 0.1 M HEPES (see Table). DNA can be solubilized in water, but it may take more effort to do so than with an alkaline solution. Typical yield is 20 - 40 µg of DNA / ml whole blood. Add an adequate amount of 8 mM NaOH or water to achieve a DNA concentration of about 0.1µg/µl. At higher concentrations, the solution is extremely viscous due to the presence of high molecular weight DNA. Alkaline solutions are neutralized by CO₂ from the air. Once a month, prepare 8 mM NaOH from a 2 - 4 M NaOH stock solution that is less than 6 months old.

QUANTITATION OF DNA AND RESULTS
Dilute an aliquot of the solubilized DNA with water, 8 mM NaOH or 1-3 mM Na₂HPO₄ and measure the A260 and A 280 of the resulting solution. A slightly alkaline solution is optimal for the spectrophotometric analysis of RNA and DNA (1). Calculate the DNA content assuming that one A260 unit equals 50 µg of double-stranded DNA/ml (2). Genomic DNA isolated with DNAzol BD has a molecular weight ranging from 40 to 100 kb and an A260/280 ratio of 1.7 - 1.9. The molecular weight of the isolated DNA is influenced by the extent of DNA shearing during the solubilization. In addition to genomic DNA, DNAzol BD also isolates small DNA fragments (down to 100 bp) which allows DNAzol BD to be used for the isolation of apoptotic DNA fragments and viral DNA (see Note 3).

NOTES and COMMENTS

1. Isolation of small amounts of DNA (<10 µg ) can be performed in the presence of Polyacryl Carrier (cat. no. PC 152) . Add 3 - 5 µl of Polyacryl Carrier to the initial lysate (Step 1) and follow the standard protocol by precipitating the DNA and carrier mix with isopropanol.
2. The isolation procedure can be interrupted and samples can be stored at the following steps. Before or after the initial centrifugation (Step 2), the DNAzol BD lysate can be stored for at least one week at room temperature, and at least one month or one year at 4C or -20C, respectively. The DNA pellet can be stored in 95% ethanol for at least one week at room temperature or for one year at 4C.
3. DNAzol BD can be used for the isolation of apoptotic DNA fragments from whole blood and viral DNA from serum. For the isolation of apoptotic fragments follow the standard protocol. For the isolation of viral DNA, substitute whole blood with an equal volume of serum and supplement the initial lysate (Step 1) with 3 - 5 µl of Polyacryl Carrier / ml of serum. Do not add more than 10 µl of Polyacryl Carrier per sample. Perform DNA precipitation using 0.5 volumes of isopropanol per one volume of DNAzol BD used for the initial lysis. Wash the DNA-carrier pellet as described in the standard protocol. Dissolve the final pellet containing viral DNA and Polyacryl Carrier in water by heating at 50C and / or vortexing.
4. DNAzol BD can be used to isolate DNA from small quantities of whole blood (< 20µl) or from dried blood on a blood filter card (approximately 5 µl per sample). The blood filters can be processed for DNA extraction and amplification in a single PCR tube. These protocols have been described (3) and reprints can be obtained by contacting MRC.

1. Wilfinger W., Mackey K. and Chomczynski P. 1997. Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques, 22, 474 - 481.
   
2. Ausubel F., Brent R., Kingston R., Moore D., Seidmann J. and Struhl K. 1990. Current Protocols in Molecular Biology, 3, A.3D.1, John Wiley & Sons, Inc. New York, NY.
   
3. Mackey K., Steinkamp A. and Chomczynski P. 1997. DNA Extraction from Small Blood Volumes and Single-Tube DNA Extraction and Amplification Using Blood Filter Cards. Molecular Biotechnology, 9, 1-5.
   
4. Chomczynski P., Wilfinger W. and Mackey K. 1998. Isolation of Genomic DNA from Human, Animal, and Plant Samples with DNAzol Reagents. Biotechnology International, 185-188.