DNeasy Blood and Tissue Kits for DNA Isolation

For DNA isolation using the QIAamp DNA Mini, or the DNeasy Blood & Tissue Kit, we recommend using carrier DNA when expected yields are below 10 ng. If possible, carrier DNAs such as poly-dA, poly-dT or poly-dA:dT should be used. Other carrier DNAs such as herring sperm DNA may interfere with subsequent PCR by binding primers nonspecifically.

Please note that poly-dA may interfere with oligo-dT primers, and, in this case, a different carrier DNA should be used. The concentration of carrier DNA should be at least 10 µg/ml. Optimal amounts need to be determined empirically for each application. The size distribution of carrier DNAs is typically in the range of 100 bp to 10 kb.

It is strongly advised to follow the recommendations for preparing sample material provided in the corresponding Protocol Sheet to ensure reliable results.

Plasma: It is recommended to perform plasma separation immediately after blood collection when using EDTA as anticoagulant to prevent the release of genomic DNA into the plasma fraction.

Urine: Because circulating cell-free DNA in non-stabilized urine samples is rapidly degraded after sample collection due to high nuclease activity, eluates may contain no DNA or exhibit low DNA concentration. Therefore, it is recommended to stabilize urine samples. Even when using stabilized urine, it is recommended to perform a centrifugation step immediately after stabilization to prevent the release of genomic DNA from cells. Alternatively, non-stabilized urine samples can be processed immediately after collection and centrifugation using ATL pretreatment and automated DNA extraction as described in the corresponding Protocol Sheet.

Yes, please follow either of the User-Developed Protocols:

• 'Acetyl Cysteine (NALC) treatment of viscous samples' (QA13). This protocol is for use with the QIAamp DNA Mini Kit.
• 'Acetyl Cysteine (NALC) treatment of viscous samples using the DNeasy Blood & Tissue Kit' (DY06).

Yes, please follow the Supplementary Protocol 'Purification of total DNA from yeast using the DNeasy Blood & Tissue Kit' (DY13).

The expected yield of genomic DNA isolated from bacteria with the DNeasy Blood & Tissue Kit or the QIAamp DNA Mini Kit is approximately 10 ug of DNA per 2x 10⁹ bacterial cells. Note that yields of genomic DNA will vary depending on bacterial strain, quality of the starting material, growing conditions, and the amount of material processed.

Yes, please follow either of the User-Developed Protocols:

• 'Isolation of DNA from soft tissues using the TissueLyser and QIAamp DNA Mini Kit' (QA31), for use with the QIAamp DNA Mini Kit
• Purification of total DNA from soft tissues using the TissueLyser and the DNeasy Blood & Tissue Kit' (DY11), for use with the DNeasy Blood & Tissue Kit

If you have optimized the lysis conditions for a specific sample type, you can lyse the sample in your lysis buffer, and follow the Supplementary Protocol 'Purification of total DNA from crude lysates using the DNeasy Blood & Tissue Kit' (DY15), or the corresponding protocol in the Appendix of the QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit Handbook.

We do not recommend using a vacuum manifold for DNA extraction of tissues using the DNeasy Blood & Tissue Kit. Viscosity of the lysates varies from sample to sample; therefore, the DNeasy mini spin columns are at great risk of clogging.

Yes, please follow the Supplementary Protocol 'Purification of total DNA from insects using the DNeasy Blood & Tissue Kit' (DY14).

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.

Yes, we have the following protocols:

• Isolation of genomic DNA from mosquitoes or other insects using the QIAGEN Genomic tip (QG06).
• Purification of total DNA from insects using the DNeasy Blood & Tissue Kit (DY14).

Efficient DNA isolation requires thorough sample disruption and digestion.

Although the QIAamp and DNeasy procedures requires no mechanical disruption of the tissue sample, the lysis time will be reduced if the sample is ground in liquid nitrogen or mechanically homogenized in advance. For mechanical homogenization, a rotor–stator homogenizer, such as the QIAGEN TissueRuptor, or a bead mill, such as the QIAGEN TissueLyser, can be used.

To improve digestion of tough tissue samples, Proteinase K incubation at 56°C can be performed overnight. DNA yields may be improved by increasing the amount of Proteinase K or by adding additional proteinase K after several hours of digestion.  

We do not recommend using a vacuum manifold for DNA extraction with the DNeasy 96 Blood & Tissue Kit because:

1. Viscosity of the lysates varies from sample to sample. Therefore, the wells of the DNeasy 96 plate are at great risk of clogging.
2. Use of vacuum manifold would result in incomplete removal of ethanol and salts after washing with Buffer AW2. This will affect the purity and yield of samples.

QIAGEN Proteinase K is stable for up to 1 year after delivery when stored at room temperature. To prolong the shelf-life of Proteinase K, storage at 2–8°C is recommended.

Yes, we have the following protocols:

• Isolation of genomic DNA from sperm using the QIAamp DNA Mini Kit; protocol 1 (QA03), long procedure
• Isolation of genomic DNA from sperm using the QIAamp DNA Mini Kit; protocol 2 (QA04), short procedure
• Purification of total DNA from animal sperm using the DNeasy Blood and Tissue kit; protocol 1 (DY02), long procedure
• Purification of total DNA from animal sperm using the DNeasy Blood and Tissue kit; protocol 2 (DY03), short procedure
• Purification of DNA from epithelial cells mixed with sperm cells using the QIAamp DNA Micro Kit (QA40).

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.).

Dedicated QIAcube kits are the RNeasy Mini QIAcube Kit, QIAamp DNA Mini QIAcube Kit, the QIAamp DNA Blood Mini QIAcube Kit, and the QIAamp Viral RNA Mini QIAcube Kit. In addition to these kits are the QIAamp PowerFecal Pro DNA QIAcube Kit, the DNeasy PowerSoil Pro QIAcube Kit using Power technology, and the DNeasy Blood & Tissue QIAcube Kit.

Yes, please follow the User-Developed Protocol 'Isolation of genomic DNA from saliva using the DNeasy Blood & Tissue Kit; spin procedure' (DY07).

Yes, please follow the Supplementary Protocol 'Purification of DNA from cultured animal cells using the DNeasy 96 Blood & Tissue Kit' (DY12).

The composition of Buffer AE is:

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

Please see the article High-throughput DNA purification with DNeasy 96 - more than just mouse tails for this data.

Always dispose of potentially biohazardous solutions according to your institution’s waste-disposal guidelines. Although the lysis and binding buffers in QIAamp, DNeasy, and RNeasy kits contain chaotropic agents that can inactivate some biohazardous material, local regulations dictate the proper way to dispose of biohazards. DO NOT add bleach or acidic solutions directly to the sample-preparation waste. Guanidine hydrochloride in the sample-preparation waste can form highly reactive compounds when combined with bleach.
Please access our Material Safety Data Sheets (MSDS) online for detailed information on the reagents for each respective kit.