Resuspending Dry Oligos
Oligonucleotides are shipped in dry form unless otherwise requested. Sometimes the dried DNA becomes dislodged from the bottom of the tube during shipping. This "loose" DNA can easily fly out of the tube when first opened, particularly as electrostatic attraction is present if the user is wearing latex gloves.
Always briefly centrifuge oligos before opening for the first time after delivery to avoid loss of the DNA pellet!
Pure, dried DNA is usually easy to dissolve in aqueous solution. Not all oligos dry identically and some can take more time to completely resuspend than others. If the oligo solution freezes while lyophilizing in the Speed-Vac the oligo will dry as a filamentous powder that appears like a fine piece of Kleenex or kimwipe. This form of DNA goes almost instantly into solution (but is prone to "fly out of the tube" as noted above). If the DNA does not freeze while drying, it can dry as a clear film coating the bottom and sides of the tube. This form of DNA will also go completely into solution but sometimes requires vigorous vortexing over a period of several minutes to ensure complete resuspension, especially if the oligo yield is large (milligram yields). Heating will speed this process. Phosphorothioate modified oligos are particularly difficult to resuspend. These oligos can be shipped in aqueous solution if requested (they are quite stable).
We recommend dissolving the stock oligo in concentrated form in TE (10 mM Tris pH 8.0, 1 mM EDTA). Alternatively, sterile dH2O can be used. DNA kept frozen in a nuclease-free environment should be stable for years. We find it convenient to initially make a freezer stock (which should be thawed relatively infrequently) at 100 µM concentration. Adding a volume of TE (µl) equal to ten times the number of nanomoles of DNA present in the tube (as noted on the spec sheet provided with the oligo) will produce a stock solution at this concentration. For example, dissolve 50 nmoles of oligo in 500 µl TE to make a stock 100 µM solution. Dilute from this stock 1:10 (in water) to make a working solution at 10 µM for use in setting up PCR reactions. Most PCR reactions use 0.1 - 0.5 µM primer. Addition of 1 µl of the 10 µM primer to a 20 µl PCR reaction will result in a final primer concentration of 0.5 µM, or 10 picomoles of oligo in 20 µl volume.
For those preferring to work in mass units, the amount of oligo present in each tube is also indicated on the spec sheet in both OD260 units and milligrams. A 20-mer primer with random base composition will have a molecular weight of ~6100 and an extinction coefficient of 196900 L/mole-cm. 1 OD260 unit of this oligo will therefore correspond to 31 µg or 5 nmoles of DNA. Dissolving 500 µg of DNA (16 ODs) in 500 µl TE will result in a stock primer of 1 µg/µl, or about a 160 µM solution (160 picomoles / µl). Again, a stock solution at this concentration will need to be diluted further before use, bearing in mind that about 10 picomoles of DNA (or about 60 ng) is sufficient for a single 20 µl PCR reaction.