QIAamp DNA Kits for DNA Extraction

Yes, please follow the Supplementary Protocol 'Purification of REPLI-g amplified DNA using the QIAamp DNA Mini Kit' (RG14).

Yes, please follow the User-Developed Protocol 'Purification of genomic DNA from cultured cells using the QIAamp DNA Micro Kit' (QA43).

Note:  The maximum amount of cells that can be used with this protocol has not been thoroughly tested.  However, we would suggest using no more than 1 x 10⁶ cells.

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.

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.

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.

• 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.

Fragments of approximately 200 bp can be isolated with good recovery. Smaller fragments can also be isolated but the recovery will be reduced with decreasing fragment lengths.

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.

The MinElute spin columns included in the following kits should be stored at 2–8°C upon arrival: AllPrep DNA/RNA Micro, EpiTect Fast DNA Bisulfite, EpiTect Fast FFPE Bisulfite, EpiTect Fast LyseAll Bisulfite, EpiTect Plus DNA Bisulfite, EpiTect Plus FFPE Bisulfite, EpiTect Plus LyseAll Bisulfite, exoRNeasy Serum/plasma Maxi, exoRNeasy Serum/Plasma Midi, GeneRead DNA FFPE, GeneRead rRNA Depletion, GeneRead Size Selection, MinElute Gel Extraction, MinElute PCR Purification, MinElute Reaction Cleanup, miRNeasy FFPE, miRNeasy Micro, miRNeasy Serum/Plasma, QIAamp DNA FFPE, QIAamp DNA Investigator, QIAamp DNA Micro, QIAamp MinElute Media, QIAamp MinElute Virus Spin, QIAamp MinElute Virus Vacuum, RNeasy FFPE, RNeasy Micro, RNeasy Plus Micro.

Short-term storage (up to 4 weeks) at room temperature (15–25°C) does not affect the performance. However, for optimal performance and quality, storage temperature should not exceed 25°C.

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.  

Protocols for automated DNA extraction from saliva stabilized with the PAXgene Saliva Collector are available for the QIAsymphony DNA Midi Kit with the QIAsymphony SP instrument or the QIAamp DNA Mini Kit with the QIAcube (Classic and Connect). 
For manual extraction, supplementary protocols are available for the QIAamp DNA Mini and Gentra Puregene Cell Kits (see resources section).

The expected DNA yields from different tissues using the QIAamp DNA Mini Kit are as follows:

DNA was purified with QIAamp Kits following standard protocols.

Using QIAGEN Genomic-tips

Protocol for DNA cleanup using Genomic-tips can be found in the 'Special Applications' section of the QIAGEN Genomic DNA Handbook.

Using QIAamp DNA Micro kit

Up to 10ug of gDNA can be cleaned using the cleanup protocol in the QIAamp DNA Micro Handbook.

Using Gentra Puregene Reagents

Gentra Puregene Handbook has protocols for removal of proteins and RNA from purified gDNA in Appendix C and Appendix D respectively.

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.

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.

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, we have the following protocols:

• Isolation of genomic DNA from compact bone using the QIAamp DNA Mini Kit (QA02)
• Isolation of genomic DNA from compact bone using the DNeasy Blood & Tissue Kit (DY01)
• Purification of genomic DNA from bones using the QIAamp DNA Micro Kit (QA39)
• Purification of archive-quality DNA from bone fragments using the Gentra Puregene Tissue Kit (PG38).

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).

Yes, we have the following protocols:

• Isolation of genomic DNA from fungi (culture and blood) using the QIAamp DNA Mini Kit (QA09).
• Isolation of genomic DNA from plants and filamentous fungi using the QIAGEN Genomic-tip (QG08).

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 User-Developed Protocol 'Detection of Bordetella pertussis DNA by PCR' (QA14). The procedure is for use with the QIAamp DNA 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, 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