The following is a list of frequently asked questions related to GenElute™-E single spin DNA and RNA purification kits.
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.
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.
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.
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.
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.
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.
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!
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.
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).
Molecular weight cutoffs (MWCOs) are always a concern when working with long DNA fragments. However, GenElute™-E technology has been shown to work with a wide range of molecular weights (as low as 65 bp and as high as 49,000 bp). Please note, some variability may be observed due to tertiary structures of nucleic acids.
The GenElute™-E Single Spin DNA CleanUp kit is not recommended for this application since the gel matrix has the potential to clog the purification resin.
GenElute™-E Single Spin purification kits work with all relevant downstream nucleic applications.
Yes, it is recommended to use the 1X Tris buffer as a blank. However, water can also be used.
Unfortunately, the columns cannot be reused. If the sample exceeds 100 µL, a new column is required. However, the spin columns are available separately as GenElute™-E Single Spin CleanUp kits. The scale for the sample load is based on the protease enzyme limitations, so those would need to be scaled and can be optimized for the sample. However, the volume loaded is the limiting factor.
This step is meant to clear the sample with an easily depletable salt. The maximum centrifugation speeds for all common microcentrifuges are all within range of performing this step without fractioning the nucleic acid into the pellet.
Different samples require different shaking speeds to keep the sample in suspension. Extending lysis times and adding a vortexing step during incubations can improve lysis for certain sample types.
The initial incubation is the optimal temperature for the SmartLyse™ enzymes. The second incubation is a heat inactivation step and will also release any partially digested proteins from the nucleic acid.
If using the cap puncher protocol, a wide-bore tip is not recommended. If a wide-bore tip is required, please do not attempt the cap puncher protocol. Typically, during the clearing step, the particulates that could clog the tip are pelleted and the supernatant is loaded. However, if a sample has a high nucleic acid content, it may be too viscous to use a gel loading tip. Standard pipet tips are recommended when performing the cap puncher protocol.
Due to centrifugal forces, oftentimes a slant is observed with the resin in the prepped column. It is recommended to load the sample vertically so that the sample disperses evenly across the resin and is not concentrated to one side.
Pipet the sample slowly and evenly so as not to greatly disturb the packed resin beads.