QIAamp DNA Blood Kits – Genomic DNA Extraction

One milliliter of healthy human blood consists of cell types in approximately the following numbers:

• Leucocytes (function: immune response) 4–7 x 106 cells
• Thrombocytes (function: wound closing) 3–4 x 108 cells 
• Erythrocytes (function O2 and CO2 transport) 5 x 109 cells

Alcohol precipitation is commonly used for concentrating, desalting, and recovering nucleic acids. Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitation of DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co-precipitation of salt that interferes with downstream applications.

Procedure

1. Adjust the salt concentration, for example, with sodium acetate (0.3 M, pH 5.2, final concentration) or ammonium acetate (2.0–2.5 M, final concentration).
2. Add 0.6–0.7 volumes of room-temperature isopropanol to the DNA solution and mix well.
3. Centrifuge the sample immediately at 10,000–15,000 x g for 15–30 min at 4°C
4. Carefully decant the supernatant without disturbing the pellet.
5. Wash the DNA pellet by adding 1–10 ml (depending on the size of the preparation) of room-temperature 70% ethanol. This removes co-precipitated salt and replaces the isopropanol with the more volatile ethanol, making the DNA easier to redissolve.
6. Centrifuge at 10,000–15,000 x g for 5–15 min at 4°C.
7. Carefully decant the supernatant without disturbing the pellet.
8. Air-dry the pellet for 5–20 min (depending on the size of the pellet).
9. Redissolve the DNA in a suitable buffer.

Tip: Use a buffer with a pH of 7.5–8.0, as DNA does not dissolve easily in acidic buffers. Often distilled water can have an acidic pH. The addition of EDTA protects the DNA from DNase digestion.

Tip: High-molecular-weight DNA, such as genomic DNA, should be redissolved very gently to avoid shearing. If the DNA pellet does not dissolve easily, heat at 55°C for 1–2 h with gentle shaking.

• Add 1/10 volume of 3 M Na-Acetate pH 5.2, and 2 to 2.5 volumes of ice-cold 100% ethanol to the DNA sample
• Mix, and store at –20°C for at least 1 h to precipitate the DNA
• Recover the precipitated DNA by centrifugation at full speed in a microcentrifuge for 15–20 min
• Pour off the ethanol and wash the pellet twice with room-temperature 70% ethanol
• Allow the DNA pellet to air-dry
• resuspend the DNA in a suitable volume of sterile TE buffer or distilled water

QIAGEN Protease is inactivated by incubation at 70°C for 15 minutes.

To our knowledge, Proteinase K cannot be completely heat-inactivated. Even when incubating at 95°C for 10 minutes, some enzymatic activity remains. This will not negatively affect the QIAamp Procedure, since the enzyme will be efficiently removed by the wash steps in the protocols.

Yes. We have shown that DNA purified with the QIAamp DNA Blood Mini Kit is stable for at least 10 years at either 2–8ºC or –20ºC. However, our data indicates that DNA stability is dependent upon elution buffer used for storage. For detailed information on this stability study see the QIAGEN News Article for the first part of the study and then the continuing study data at the 10 year mark.   

Yes. Please follow the Blood and Body Fluid Spin Protocol in the QIAamp DNA Mini and QIAamp DNA Blood Mini Kit Handbook. No more than 5 x 10⁶ cells should be used. When using the Midi or Maxi format, please follow the protocol for Isolation of DNA using the QIAamp DNA Blood Midi or Maxi Kit in the QIAamp Blood Midi and QIAamp Blood Maxi Handbook. The maximum number of cells to use is 2 x 10⁷ and 1 x 10⁸, respectively.

Tissue is more difficult to lyse than cells in a blood sample. Therefore an additional incubation step in buffer ATL containing Proteinase K is included in the Tissue Protocol to achieve complete sample lysis. Blood cells will directly lyse by incubation in Buffer AL containing QIAGEN Protease. Buffer AL is required in both protocols to ensure appropriate conditions for DNA binding to the silica membrane. For further details on the protocols, please refer to the QIAamp DNA Mini and QIAamp DNA Blood Mini Kit Handbook.

According to an Interview with Professor Dennis Lo published in QIAGEN News Molecular Diagnostics, Issue No. 5, 2002, healthy individuals have about 500-1000 genome equivalents (DNA) per ml serum/plasma.

For free-circulating DNA in plasma, the concentration can range from 1–100 ng/ml in healthy individuals.

Small amounts of RNA and DNA may be difficult to measure spectrophotometrically. Fluorometric measurements, or quantitative RT-PCR and PCR are more sensitive and accurate methods to quantify low amounts of RNA or DNA.

Fluorometric measurements are carried out using nucleic acid binding dyes, such as RiboGreen® RNA Quantitation Reagent for RNA, and PicoGreen® DNA Quantitation Reagent for DNA (Molecular Probes, Inc.).

The composition of Buffer AE is:

• 10 mM Tris-Cl
• 0.5 mM EDTA; pH 9.0.

EDTA chelates divalent cations which are required for nuclease activity. While the genomic DNA (gDNA) extracted using QIAGEN products, should not have any nuclease activity, it is possible to introduce nucleases during repeated long-term access of the DNA. EDTA helps to prevent any nuclease activity introduced after the genomic DNA extraction procedures.

However, if the gDNA is stored frozen at -20oC or -80oC, nuclease activity is much reduced. It may be possible to leave EDTA out of the storage buffer without negative consequences when samples are kept under these conditions, and when repeated freeze-thaw cycles are avoided.

We do recommend however that gDNA be stored in a neutral to a slightly basic buffered solution (e.g. 10 mM Tris-Cl pH 8.5 to 9.0) to prevent DNA degradation by acid hydrolysis. Note that deionized water mostly has an acidic pH.

QIAGEN Proteinase K is a subtilisin-type protease, which cleaves at the carboxyl side of hydrophobic, aliphatic and aromatic amino acids. It is particularly suitable for short digestion times. It possesses a high specific activity over a wide range of temperatures and pH values with substantially increased activity at higher temperature. Soluble calcium is not essential for enzymatic activity. This means that EDTA, which is used to inhibit Mg2+-dependent enzymes such as nucleases, will not inhibit Proteinase K activity.

QIAGEN Protease is a broad-specificity Serine protease with high activity, cleaving preferentially at neutral and acidic residues. It is an economical alternative to Proteinase K for isolation of native DNA and RNA from a variety of samples.

Yes, please follow the User-Developed Protocol 'Detection of HBV DNA by PCR' (QA15). The procedure is for use with the QIAamp DNA Blood Mini Kit.  Please contact Technical Service for this protocol.

Yes, we have the following protocols:

• Isolation of genomic DNA from saliva and mouthwash using the QIAamp DNA Mini Kit; vacuum procedure (QA19v)
• Isolation of genomic DNA from saliva and mouthwash using the QIAamp DNA Mini Kit; spin procedure (QA19s)
• Isolation of genomic DNA from saliva using the DNeasy Blood & Tissue Kit; spin procedure (DY07)

Yes, we please follow the Supplementary Protocol 'Isolation of genomic DNA from dried blood spots using the QIAamp 96 DNA Blood Kit' (QA22).  Please contact Technical Service for this protocol.

Yes, please follow the User-Developed Protocol 'Isolation of bacterial DNA from soil using the QIAamp DNA Stool Mini Kit and QIAamp DNA Blood Midi Kit' (QA28).