Genomic DNA Purification Support—Getting Started

The ratio of absorbance at 260 to the absorbance at 280 nm (A260/A280) is typically used to measure purity of the sample. For DNA, the ideal ratio is 1.8, but could be in the range 1.7–1.9. The A260/A230 ratio is also used to determine if contamination is present. The ideal ratio for DNA is 1.8–2.0. Purity of DNA can also be examined by gel analysis. For genomic DNA, look for high average fragment size.

We would recommend using our ChargeSwitch® technology, which demonstrates high sensitivity for purifying DNA from small or degraded samples. 

Please see the following web page for a comparison of kits that can be used to isolate genomic DNA from blood.

Yes, we offer our ChargeSwitch® Forensic DNA Purification Kit. Please read more about DNA extraction here.

We offer several kits specifically designed to isolate plant DNA depending on the amount of starting material and chemistry you are interested in using. Please use this link for a comparison of our kits available for plant DNA extraction.

We would recommend using our PureLink® gDNA Mini Kit or our ChargeSwitch® gDNA Mini Bacteria Kit (Cat. No. CS11301), which can isolate both gram-positive and gram-negative bacteria. No centrifugation or filtration steps are necessary using this simple one-tube protocol. Read more about bacterial DNA extraction here.

We would recommend using one of our ChargeSwitch® gDNA Buccal Cell Kits, which were designed to isolate high-quality genomic DNA from buccal samples. We also offer normalized kits, which will normalize isolated DNA concentration from sample to sample.

We offer three kits: RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE, MagMAX™ FFPE Total Nucleic Acid Isolation Kit, and the MagMAX™ FFPE DNA Isolation Kit. Read more about the differences between the three here.

There are a number of factors that can impact the overall quality and yield of DNA isolated from FFPE tissues. Here are recommendations to address several key factors:

1. Upstream tissue procurement and tissue specimen preparation—if possible, tissues should be fixed within one hour of surgical resection. The optimal fixation time is 12–24 hours using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
2. Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungi, insects, etc.).
3. Tissue type, size, and amount being used for DNA isolation—the recommended tissue thickness is 10–20 µm. The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50–300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
4. Excessive amount of paraffin used for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, a more rigorous 37–55°C treatment can be performed for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.

Read more about extraction of nucleic acids from FFPE samples here.

Here is a list of DNA/RNA Purification replacement buffers we offer as standalone.

Yes. However, the yield from frozen blood is significantly lower than the yield from fresh blood.

The JetQuick Blood and Cell Culture DNA Maxiprep Kit can be used for blood samples >10 ml. The manual offers a protocol for the concentration of the sample first.

No. The JetQuick Blood and Cell Culture DNA Midiprep Kit and Maxiprep Kit are not validated for tissue samples.

Yes. However, the yield from frozen blood is significantly lower than the yield from fresh blood.

Yes, single-stranded DNA will separate with the DNA phase.

DNAzol® Reagent is designed to use with tissues, cells or blood. It includes a guanidine-detergent lysing solution that selectively precipitates DNA from a cell lysate. It can isolate genomic DNA from 50 mg of tissue to 1 x 10^7 to 3 x 10^7 cells with 1 mL of DNAzol® Reagent. DNAzol® BD Reagent was specifically formulated for the isolation of genomic DNA from blood. It can be used to isolate genomic DNA from 0.5 mL whole blood with 1 mL of reagent. Plant DNAzol® Reagent was specifically formulated for isolation of genomic DNA from plants. It can isolate genomic DNA from 0.1 g of plant tissue with 0.3 mL of reagent.

We recommend storing DNAzol® Reagent at room temperature (stable for 1 year). It can also be stored at 4°C. Storage for 1 week at 37°C showed no deleterious effects. The DNAzol® lysate (homogenate) can be stored 1 month at 15–30°C; after 10 months at 4°C or –20°C, the DNAzol® lysate (homogenate) has yielded high molecular weight genomic DNA, which can be completely digested with restriction enzymes and works well in PCR. During washes, DNA can be stored in 95% EtOH for at least one week at 15°C to 30°C or for three months at ~4°C. DNA can be stored in DNAzol® Reagent for one month at room temperature or 10 months at 4°C.

To reduce/eliminate the amount of RNA in a sample, treat the genomic DNA with RNase A at 1 µg/mL.

Trypsinization is not needed. Simply add 1 mL of DNAzol® Reagent per 10 cm2 of the culture plate area. Rock the plate back and forth and pipet the contents into a tube for ethanol precipitation of genomic DNA. If you want to trypsinize to count your cells, pellet the cells that will be used for DNA isolation after trypsinization. Remove the supernatant completely by aspiration, then add DNAzol® Reagent to the cell pellet. Pipet 3–4 times to lyse the cells completely before proceeding to the centrifugation step.

The DNA isolated is actually total DNA, so plasmid DNA will be isolated along with genomic DNA. The mitochondrial genome is similar to a plasmid and can be isolated using DNAzol® Reagent. The 1 minute room temperature incubation in ethanol before centrifugation should be extended to 5–10 minutes for maximum recovery.

GeneCatcher™ technology is based on magnetic bead-based gDNA purification. It is designed to extract gDNA from human blood without the use of centrifugation. The GeneCatcher™ kits have also been designed and optimized for scalable and automated genomic DNA isolation from blood samples using the Tecan Freedom EVO liquid handling platform. View the workflow here.

Yes. The kits work with EDTA, citrate, and heparin. Although heparin does not affect purification when using GeneCatcher™ gDNA Blood Kits, it is not recommended for use as an anticoagulant, as trace amounts are powerful PCR inhibitors.

Yes, it can. Treat the buffy coat sample with the volume of reagents that you would have used for the whole blood volume from which the buffy coat originated. You may have to divide the sample into multiple aliquots for processing if the original blood volume was greater than 10 mL.

Please use this link for a comparison of the two systems.

The MagMAX™ Express-96 plates, deep well plates (squared wells), and PCR plates (skirted) can be used with the instrument. Each plate type has an optimized MagMAX™ Express-96 head and tips. Of the three plates, the MagMAX™ Express-96 plate can be used with all three kinds of magnets. The volume of the deep well plates has to be a minimum of 2.2 mL (2.0 mL capped) with squared wells.

Yes. Both deep well plates and MagMAX™ Express-96 plates can be used during the same run. Therefore, it is possible to start the processing by using larger volumes (in a deep well plate) and elute the purified sample to a smaller volume (in a MagMAX™ Express-96 plate).

We offer a complete MagMAX™ Express-96 system called the MagMAX™ Express-96 Magnetic Particle Processor for the purification and processing of proteins, DNA, RNA, and cells in a 96-well format. The processor handles particles automatically according to the preloaded purification protocols.

The MagMAX™ Express-96 magnets are made of material that is very stable. The magnetic field will not get weaker. However, extreme mechanical force or heating can cause damage to the magnets.

Note: It is very important to keep the MagMAX™ Express-96 heads away from each other and other magnets at all times. Clashing of the magnets together causes serious damage to the magnets.

The MagMAX™ Express-96 system should not be kept in close proximity to magnetic tapes, computer discs, or other magnetic storage systems, such as credit cards, as these can be damaged by the strong magnetic field of the MagMAX™ Express-96 heads. Do not hold the MagMAX™ Express-96 heads close to a PC display, since this may cause damage to the display. Do not use metal tools when handling MagMAX™ Express-96 heads.

Warning: This product contains very strong permanent magnets. People wearing a pacemaker or metallic prosthesis should not use this product. A pacemaker or prosthesis may be affected or damaged if it comes in close contact with a strong magnetic field.

The heating block is located inside the instrument and can be used automatically during the protocol. Both MagMAX™ Express-96 plates and PCR plates (skirted) can be heated using specially designed, interchangeable heating blocks. Any number of heating steps can be added to the protocol. During the protocol, when the protocol enters the heating step, the plate is automatically moved to the dedicated heating position for heating. After the heating step, the protocol continues automatically to the next step.

Note: No cooling steps can be added.

The heating block warms up about 10°C per minute, so it will take about 6 minutes.

We strongly recommend that you keep the specified volumes within the defined limits to avoid spillover of the reagents.

Mixing with the tip comb is designed to occur below the liquid surface. This prevents aerosols and splashing from occurring when using the MagMAX™ Express instrument.

There are 2 rows of 12 magnetic heads each, and 2 plates for loading.

Deep well plates are recommended. Use a deep well configuration for larger sample inputs (up to 400 μL).

No, unfortunately not.

Yes, you can use 0.1 mM EDTA as an alternative (though beads may pellet slower).

It is a personal preference as to which magnetic stand to purchase. Cat. No. AM10027 can be easier if using a pipettor for liquid handling, as beads clump to the side, Cat. No. AM10050 if using a robotic liquid handler, as beads clump in a ring.

Table 1 of the manual lists all the different plants we have tested with the kit and the approximate DNA yields to expect.

You can find the .bdz files on the product page.

If higher DNA concentration is needed, the elution volume can be reduced to 50 µL when using the KingFisher Flex instrument. However, we have not tested this and the script may have to be changed accordingly.

PVP adsorbs polyphenols, removing them from the final eluate. DTT, a reducing agent, protects the DNA from degradation by preventing the oxidation of polyphenols. We generally recommend adding PVP for woody, lignified, or polyphenol samples (branches, twigs, needles, and wax-coated leaves). DTT can be helpful when working with seeds. Some seeds (e.g., seeds with a pesticide coat) do benefit from addition of PVP to Lysis Buffer A. If DTT supplementation to Lysis Buffer A was already attempted and has not helped, try again with PVP instead.

Yes, the iPrep™ PureLink® Total RNA kit will do this. When prompted on screen, you could select the "No DNase" option. This will result in collection of all nucleic acid present in the sample. However, this kit was not optimized to purify DNA, so there would be preferential binding for RNA over DNA due to the GITC in the lysis/binding buffer.

The iPrep™ Purification Instrument allows for automated purification of nucleic acids from up to 13 samples (12 samples and 1 positive control). You will need to purchase an iPrep™ Kit, which contains the sample preparation reagents and reagents, as well as the iPrep™ Protocol Card, which is preprogrammed with the purification protocol that directs the volume of reagents used and incubation time.

The iPrep™ Purification Instrument has been discontinued but we still offer iPrep™ purification kits and pre-programmed iPrep™ protocol cards to go along with the instrument.

The size range is between 100 bp and 12 kb. Our kit comes with two buffers, Binding Buffer (B2) and Binding Buffer HC (B3). The Binding Buffer HC (B3) eliminates primer-dimers and short failed PCR products that are smaller than 300 bp.

The micro kit uses the same buffers (except the HC buffer) as the original kit, but a different (micro) column. The combo kit also contains the same spin column, but has an extra buffer, Gel Solubilization Buffer (L3), for gel extraction.

The size cut-off is 100 bp to 12 kb.

The buffers and procedure are different for the PureLink® PCR Purification Kit and PureLink® Gel Purification Kit. Therefore, you can swap the column, but the right buffer and protocol must be used.

Yes, you can use our kit to clean up restriction digested plasmid DNA.

Yes, you can purify ANY dsDNA sample with a size range of 100 bp to 12 kb from liquid samples with the PureLink® Quick PCR Clean-Up Kit. Supercoiled DNA can be purified as well, but some nicking should be expected.

The PureLink® kit is based on a spin column format, while the ChargeSwitch® kit is based on magnetic bead-based technology. In low pH conditions, the beads have a positive charge and bind the negatively charged nucleic acid backbone.

Yes, please view this web page for a comparison of sequencing results from amplicons purified using the ChargeSwitch®-Pro PCR Clean-Up Kit and the QIAquick® PCR Purification kit.

Unfortunately, we do not offer crystal violet as a standalone product. Rather, this reagent is contained in the TOPO XL kits as well as the S.N.A.P.™ UV-Free Gel Purification Kit (Cat. No. K200025).

Yes, both TAE and TBE agarose gels are compatible with our gel extraction kits.

DNA fragments ranging in size from 40 bp to 10 kb can be purified with the PureLink® Quick Gel Extraction Kit. However, recovery of fragments less than 80 bp will be reduced by 50% or more. For single-stranded DNA and RNA, the lower limit is about 200 nt.

Yes, water may be used, but please ensure that the water is clean and the pH of the water is between 7.5 and 8.5.

Both regular and low-melting agaroses can be used. When the agarose concentration is above 2%, use 600 μL of solubilization buffer for each 100 mg of gel.