GenElute™-E Single Spin DNA and RNA Purification Kits FAQs

The following is a list of frequently asked questions related to GenElute™-E single spin DNA and RNA purification kits.

Technology FAQs

Application FAQs

Protocol FAQs

GenElute-E Single Spin DNA and RNA purification kits use negative chromatography to isolate nucleic acids. Can you explain how this approach simplifies workflows?

Instead of optimizing the bind, wash, and release steps in conventional silica-based spin purification preps, the technology focuses on a separation by performing a single step fractionation based on the size of the biomolecules, which results in depleted impurities.

What’s so special about SmartLyse enzymes?

Targeted SmartLyse™ enzymes reduce lysis time and enable a more efficient workflow, releasing the scientist from tedious tube handling by eliminating bind and wash steps.

Often when using silica columns for purification, a scientist can elute with a smaller volume to have a more concentrated sample, or elute twice for greater yield. How does this technology differ?

When eluting with a smaller volume using silica, a higher concentration can be achieved. However, residual salts and ethanol needed to bind and wash the sample will also be present at higher concentration. With negative chromatography, the volume you load is essentially the volume you recover (typically ~80-100 µL), which is usually more concentrated than silica isolated nucleic acid samples. With silica, a second elution usually releases more process contaminants and smaller fragmented nucleic acid, which can lead to inaccurate quantitation and result in an overestimation of yield.

How does GenElute-E technology improve the accuracy of nucleic acid quantitation?

There are no process contaminants introduced when using negative chromatography for purification, and more full-length nucleic acids are recovered by reducing centrifugation steps. This improves the accuracy of quantitation leading into downstream applications and eliminates contaminants that can interfere with the downstream application itself, making for more robust experiments.

What other advantages does GenElute-E offer researchers?

Eliminating bind and wash steps is a more sustainable approach of isolating nucleic acids, reducing the amount of plastic tubes and flow-through chemical waste. This makes GenElute™-E a sustainable, green chemistry option that is more beneficial for everyone.

I am using a finnicky amplification enzyme (polymerase). Does this technology eliminate Mg+2 ions from my preps?

It does! GenElute™-E single spin purification technology depletes ions, allowing for more robust downstream enzymatic reactions by ensuring that the concentration of components of the enzymatic buffer is better optimized without any “surprise” inhibitors.

What is the composition of the lysis buffer and clearing buffer after flowing through the resin?

The presence of EDTA, SDS, or excess salt can affect my PCR/ sequencing reaction...

The lysis buffer information is proprietary, but we can say it is free of chaotropic salts. The resins are desalting resins so EDTA, SDS, and salts are depleted. That’s the beauty of the technology!

Does the technology introduce any bias into the sample?

GenElute™-E does not introduce biases that some “bind-wash-elute” technologies can add because the technology separates by size, rather than by what binds and what is released.

Do we know how stable the purified DNA is through several freeze-thaw cycles?

This will fluctuate due to sample variability (sample collection, concentration, fragment length, sequence [GC content], storage before isolation, etc.). However, the sample is buffer exchanged into a standard storage buffer that is included in the kit (1X TE).

Firstly, regarding shearing and reducing shear damage associated with multiple spins on a spin column - is this relevant for RNA? Secondly, how does the negative chromatography column exclude large negatively charged proteins/glycoproteins when you want to extract nucleic acid (specifically, RNA)?

• RNA extractions are prone to fragment degradation, typically from enzymatic digestions within the sample (RNases). Therefore, it important to isolate the nucleic acid from these enzymes as quickly as possible. In a typical bind-wash-elute spin prep procedure, sample shearing occurs due to multiple centrifugation steps as well as the process of binding and releasing the biomolecule from a membrane or bead/resin. Removing these process variables from the workflow of RNA isolation allows greater control of the final sample fragment quality. However, some downstream applications do not need this level of control since they are less sensitive to these degradations. Controls do help, especially for samples with lower yields, and can aid in troubleshooting if downstream processes fail.
• There are many chromatography methods that take advantage of differences in the biomolecules for separation, such as ions/charge for silica. “Negative chromatography” works by size exclusion, allowing the larger molecular weight products to be collected first. We can totally understand the intuitive equating our use of the word “negative” and charge! Proteins, which are digested by the SmartLyse™ enzymes, will flow more slowly through the resin matrix than the RNA molecules, and will remain within the column post-centrifugation. Quick trick: If you wish to collect these digested protein fragments along with the other smaller molecular weight biomolecules, you can spin the column after purification at a higher speed to collect to the slower moving fraction.