Great turn around time on my 80 exome project!
If using very short DNA samples, salt should be added to the eluted product. The salt concentration should vary depending on the downstream applications; for most applications we suggest using a final concentration of 10 mM Tris (pH 7.5-8), 5 mM NaCl, 0.1 mM EDTA. We also recommend not heating short DNA samples before adjusting salt concentration to avoid denaturing of the sample.
We only use sterile deionize water in all steps during swelling, washing and final suspension of the gel matrix. However, due to the manufacturing process of the resin that we use in the gel matrix, a small amount of salt (approximately 3 mM) leaches from the gel and is present in the final elution. This amount of salt does not affect any standard downstream application
We cannot sterilize the cartridges after filling them and we do not include preservatives in the gel matrix. However, we use sterile water to swollen and wash the gel matrix; we operate in clean hoods and our manufacturing facilities have clean air filters. We test a sample of our cartridges for contamination as part of the Quality Control.
We cannot guarantee that the cartridges are RNase free, since we do not test for it in Quality Control. However, we operate in clean hoods and our manufacturing facilities have clean air filters, lowering the probabilities of RNase contamination in the cartridges. Some of our customers are using the DNA clean up cartridges for RNA work and they have not experienced any problems.
It depends on how clean the DNA is and how sensitive the mammalian cells are. We have achieved higher than 80% transfection efficiency in 293 HEK cells using miniprep DNA passed through Edge DNA clean up cartridges. The cartridges will remove salt and other small molecules, but they will not remove all endotoxins.
The cartridges will remove all salt and buffers. They will not remove enzymes. If enzyme removal is important, reactions should be heat inactivated prior to passing them through the cartridge (refer to enzyme manufacturer for inactivation conditions). If the enzymes cannot be heat inactivated, we recommend using Edge QuickStep® PCR clean up kit.
They will remove salt, dNTPs and some of the oligo (depending on the length and mass of the oligo and the volume loaded). It will not remove DNA-polymerase or other proteins. To clean up PCR reactions we recommend Edge QuickStep® PCR clean up kit.
For small sample volumes (lower than 20µl) higher concentration of the sample can be achieved as follows: pre-spin the cartridge at 750 x g for 3 minutes, transfer the cartridge to a clean tube, load sample onto gel matrix and spin the cartridge at 750 x g for 2 minutes. These conditions will also result in better removal of contaminants for larger volumes (50 or 100 µl). However, these spinning conditions will result in lower recovery of total DNA (the total DNA recovered is lower, the volume is much lower, so the concentration is higher).
No. Doing so will contaminate the sample with salt and other small molecules. We recommend 50µl to achieve best results. However, 100 µl load will result in small contaminations (for example, salt removal of 5M NaCl will be of 95%) that will not affect most downstream applications. To achieve better purification at 100 µl volume loaded, we recommend to prespin 3 minutes at 750 x g, transfer the cartridge to a clean tube, load the sample, and then spin 2 minutes at 750 x g. If volumes are larger than 100 µl, we recommend splitting the sample and use two different DNA clean up cartridges.
Check the spinning conditions and make sure that the rpm has been properly calculated. The presence of highly hydrophilic molecules in the reaction (such as PEG) will affect the performance of the cartridge, resulting in higher elution volume.
Check the spinning conditions and make sure that the rpm has been properly calculated. Check storage conditions and make sure that the cartridges and bag were properly closed if stored at room temperature for a short time. Cartridges should be at 4°C for long-term storage.
Spinning conditions vary depending on the volume loaded in the cartridge. For volumes lower than 20 µl, pre-spin the cartridge at 850 x g for 3 minutes, transfer the cartridge to a clean tube, load the sample onto the cartridge and spin at 850 x g for 3 minutes. For volumes equal or larger than 20 µl, pre-spin the cartridge at 750 x g for 2 minutes, transfer the cartridge to a clean tube, load the sample onto the cartridge and spin at 750 x g for 2 minutes.
The crack is a normal product of the manufacturing process of the cartridges and does not affect their performance as long as the samples are loaded properly. Load samples drop-wise to the center of the matrix, taking care to avoid adding sample directly into the crack.