Cleaning up dirty DNA

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QIAGEN is cleaning up… DNA and RNA that is. With the addition of the DNeasy and RNeasy PowerClean Pro Cleanup Kits to our portfolio, we thought it would be a great time to discuss how cleanup kits work and when it is appropriate to use one. Cleanup kits take dirty nucleic acids and remove contaminants that could interfere with downstream applications. Enzyme-dependent applications like restriction digests, ligations, PCR amplification and sequencing all require ultra-clean DNA and RNA to get the best results. Choosing the correct cleanup kit for your needs depends on which substances you want to remove. But what if you’re not sure what should be removed? Usually, these reaction-killing inhibitors are remnants either from a previous enzymatic reaction or the organism/environment that the DNA/RNA was extracted from. Our cleanup kits can be divided into two types based on which of these is the contamination source.

Our fastest and simplest kits are those that are designed to remove proteins, primers, dNTPs and salts typically left over after an enzymatic reaction; for example, PCR amplification or a restriction digest. We have three different cleanup kits that will remove these simple proteins and salts: QIAquick PCR Purification Kit, QIAquick Gel Extraction Kit and QIAEX II Gel Extraction Kit. If DNA is suspended in solution, any of these kits are appropriate for use. However, some slight differences do exist. Both QIAquick PCR Purification Kit and QIAquick Gel Extraction Kit are silica spin column-based kits. Consequently, they both have a DNA binding capacity of 10 mg per prep and will work with DNA up to 10 kb. QIAquick PCR Purification Kit will clean up DNA of 100 bp – 10 kb, and QIAquick Gel Extraction Kit will clean up DNA of 70 bp – 10 kb.

Figure 1. QIAquick spin tubes.

Figure 1. QIAquick spin tubes.

The QIAEX II Gel Extraction Kit uses a silica suspension, instead of a spin column to bind the DNA. This kit has a lower and upper DNA size limit of 40 bp – 50 kb. However, because the volume of silica can be increased or decreased as needed, it can be scaled for any quantity of DNA. The other advantage of QIAEX II Gel Extraction Kit is the option to elute the DNA in a small volume. So, it is ideal if you want to concentrate your DNA as well as clean it up. The silica spin-filter based kits have a minimum elution volume of 50 µl.

In addition to DNA in solution, the QIAquick Gel Extraction Kit and QIAEX II Gel Extraction Kit also have protocols for cleaning up DNA from an agarose gel. Since QIAEX II Gel Extraction Kit uses a silica suspension, it can accommodate any size slice of agarose gel. On the other hand, the QIAquick Gel Extraction Kit should only be used with up to 400 mg of agarose, otherwise, it might clog the spin column.

What if your dirty DNA/RNA sample contains contaminants other than proteins, primers and salts? The DNeasy PowerClean Pro Cleanup and RNeasy PowerClean Pro Cleanup Kits are the answer. These kits feature streamlined protocols that enable DNA or RNA cleanup in just 7 minutes. Inhibitors are removed using our Inhibitor Removal Technology (IRT) process for removing difficult substances that are from sources like plants, soil, feces or blood. Our original DNeasy PowerClean Cleanup Kit is recommended for removal of inhibitors found in crude heparin and heparin products.

Some common examples of contaminants, along with their typical sources include:

•  Humic acids (from soil, compost)
•  Polysaccharides (plants, undigested contents in the rumen, cecum, feces and drugs)
•  Polyphenols (plants)
•  Melanin (hair, skin and eye tissue)
•  Bile salts (feces)
•  Heme, EDTA, charcoal (blood)

Do you have a lot of samples to clean up? We have two options available. The MagAttract PowerClean DNA Kit is a high-throughput, hand-free version of the DNeasy PowerClean Pro Cleanup Kit. It contains the same Inhibitor Removal Technology and ClearMag magnetic particle technology, enabling it to be run on a KingFisher™ Flex or Duo. The UltraClean 96 PCR Cleanup Kit is a high-throughput option that is similar to the QIAquick PCR Purification Kit. It includes a 96-well silica spin filer plate that is ideal for processing large numbers of samples without a robot.

Selecting the right cleanup kit for your needs shouldn’t have to be complicated. It is important to remember that all our cleanup kits will remove proteins, primers and salts. However, only those containing Inhibitor Removal Technology will remove environmental contaminants. We’ve put the most important information in the table below so that you can easily select the perfect cleanup solution with time to spare.

Table 1. Cleanup solutions at a glance.

Cleanup Kit Binding matrix Size range Gel Solution Capacity Inhibitors Throughput
DNeasy PowerClean Pro Cleanup Kit Silica column 100 bp – 50 kb X 20 µg Environmental Low
QIAquick PCR Purification Kit Silica column 100 bp – 10 kb X 10 µg Salts, proteins Low
QIAquick Gel Extraction Kit Silica column 70 bp – 10 kb X X 10 µg Salts, proteins Low
QIAEX II Gel Extraction Kit Silica suspension 40 bp – 50 kb X X Scalable Salts, proteins Low
RNeasy PowerClean Pro Cleanup Kit Silica column 100 bp – 50 kb X 40 µg Environmental Low
UltraClean 96 PCR Cleanup Kit Silica column (plate) 60 bp – 10 kb X 20 µg Salts, protein High
MagAttract PowerClean DNA Kit ClearMag magnetic particles 60 bp – 50 kb X 500 µg per 20 µl of beads Environmental High, hands-free

Trademarks: KingFisher™ is a trademark of Thermo Scientific.

Authors: This post was originally published on mobio.com, and has been updated and modified by Heather Martinez and Miranda Hanson-Baseler.

This article was compiled from the contributions of multiple authors. Please see the end of the post for details.

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