Genomic DNA Purification Support—Getting Started
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Genomic DNA Isolation Kits
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.
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:
- 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.
- 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.).
- 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.
- 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.
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.
The phenol phase and interphase can be stored at 4°C overnight. Samples can also be stored in the washing solution (0.1 M sodium citrate in 10% ethanol) for at least a couple hours. The samples can also be suspended in 75% ethanol at 4°C for several months.
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.
PCR Product Clean-up
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.
Agarose Gel Extraction
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.
Specialized DNA Purification
Yes, the PureLink Microbiome DNA Purification Kit has been successfully used for isolation of microbial DNA from human, mouse, rat, horse, cow, dog, cat, and other types of stool/feces. Despite the fact that these samples are very different in terms of consistency, presence of miscellaneous food debris, composition of the microbial community, various inhibitors present, and other variables, the kit enabled fast and efficient isolation of substantial amounts of highly clean microbial DNA.
If fresh stool, body fluids, swabs, and other samples cannot be processed, freezing the samples at -20 degrees C or -80 degrees C is the best way to preserve them. We recommend keeping freeze/thaw cycles to a minimum. For many samples, storage at 4 degrees C for up to one week is also fine. Please note, use of commercially available DNA stabilizing reagents can negatively impact the downstream nucleic acid isolation, or make the workflow more challenging or lengthy.
We simplified the protocol for those scientists who are only interested in human DNA isolation from stool, as mammalian cells are easy to lyse and DNA isolation can be performed faster. Microbes have thicker cell membranes of a different composition, and are generally more difficult to lyse. Thus, separate protocols, with additional steps to ensure efficient disruption for isolation of microbial DNA, were developed.
Yes, when using microbial DNA protocol with PureLink Microbiome DNA Purification Kit, you will get both host and microbial DNA. The bulk of DNA in human stool is of microbial origin. Depending on the sample, we roughly estimate that approximately 10-20% of the total DNA isolated is host DNA while the remaining 80-90% is microbial DNA.
The PureLink Microbiome DNA Purification Kit was developed to be used with a wide range of sample types including stool, soil, urine, saliva, swabs, and other samples. In addition to testing bacterial samples, we have also tested fungi samples. Specialized beads in the kit efficiently break open cell walls and membranes with the help of heat and chemical lysis. This triple lysis approach works great for tough-to-lyse species such as fungi. Additionally, the stool-derived inhibitors are rapidly eliminated with a novel clean up buffer, resulting in DNA that is compatible with any type of downstream analysis.
Yes, the PureLink Microbiome DNA Purification Kit works for swabs samples. However this kit does not come with swabs. You will find some suggested swab part numbers that we have tested with this kit in swab protocol on the product page.
We tested 3 liquid versions of transport media: Amies, Stuart, and Cary Blair, with all three being compatible with the PureLink Microbiome DNA Purification kit.
For highly pure samples, both fluorometers (such as the Qubit Flourometric Fluorometer) and spectrophotometers (such as NanoDrop One Spectrophotometer) are successfully utilized and give very similar readings. However, when dealing with DNA or RNA samples that have high levels of contaminants, such as salts or protein, the Qubit Fluorometer provides a more accurate reading, as this instrument uses dyes that are very specific to the analyte of interest. We have seen some challenging microbiome and FFPE samples where the Qubit Fluorometer readings were up to 2 times lower compared to the NanoDrop spectrophotometer, which is overestimating DNA concentrations. More and more laboratories are use both instruments side by side, as they provide complimentary information: the Qubit fluorometer is more accurate and specific in terms of determining nucleic acid concentration, while the NanoDrop spectrophotometer provides a valuable information regarding the purity (ie., A260/280, A260/230).
For Research Use Only. Not for use in diagnostic procedures.