Most Frequently Asked Technical Questions

1. How do I dissolve my oligo?

Oligonucleotides may be dissolved either in sterile water or in buffer. We recommend dissolving and then aliquoting your stock solution into multiple tubes. Keep a working stock at 4 degrees Centigrade (C). Lyophilize (dry) the remaining aliquots. Store the dried oligonucleotide pellets at -70 degrees C. Oligonucleotides are the most stable when stored at -70 degrees C as a dried pellet. The liquid working stock is generally stable for up to a week when stored at 4 degrees C. The time can be shorter or longer depending upon the oligo backbone and the care taken to avoid contamination of the DNA or RNA.

2. How do I store oligos properly?

Even though it is convenient, we do not recommend storing oligonucleotides (in solution) at -20 degrees C. Repeated cycles of freezing and thawing can result in significant degradation of an oligonucleotide. Freezing and thawing occurs intentionally when an oligonucleotide stock is removed from the freezer and then returned after use. Unfortunately, unintentional cycles of freeze/thaw may also occur. The most common causes are frost-free freezers and temperature fluctuations in the freezer compartment. Changes in the storage temperature are commonly a result of frequent opening of the compartment. This is a particular problem for freezers used in common by several researchers.

3. How stable are oligonucleotides at room temperature?

Lyophilized oligonucleotides are stable at room temperature. While we have not tested long-term storage at room temperature, short term stability is quite good. That is the reason we ship oligonucleotides dry. If you are not ready to use your oligonucleotide when received, it should be stored at -70 degrees C until needed.

4. How do I determine the final yield (amount) of the oligonucleotide?

The computer analysis sheet, provided for each oligonucleotide, gives two conversion factors. The first is nanomoles per OD unit. The second is micrograms per OD unit. (An OD unit is defined as the optical density measured at 260 nanometers.) To find final yield in nanomoles: multiply the "Total A260" (given on the analysis sheet) by the nanomoles per OD conversion factor. The result is total nanomoles yield.

To find final yield in micrograms: multiply the "Total A260" (given on the analysis sheet) by the micrograms per OD conversion factor. The result is total micrograms yield.

5. I ordered a 50 nM synthesis of oligonucleotide and did not get 50 nM of material. Why?

It has become industry standard to assign synthesis scales based on the amount of starting material for a synthesis. (For example, a 50 nanomole scale means that there is 50 nanomole of the initial base coupled to the solid support used for synthesis.) Of the starting material, a substantial proportion may be unavailable for use due to chemical modification or steric problems. The actual yield should be calculated as described above.

6. I received my oligonucleotide and the tube looks empty, or I have received several oligonucleotides, all of the same scale synthesis, and the amount of sample in each tube looks uneven. Why?

The physical appearance of the dried oligonucleotide can differ due to small variations that normally occur in the drying process. Oligonucleotide appearance in the sample tubes may range from a small, glassy pellet to a relatively large white, fluffy pellet. Most samples have a white fluffy pellet. All sample tubes should be microfuged before opening, to prevent loss of any sample that may have been dispersed in the tube during shipping.

7. How can I make a concentrated stock solution?

Normally, oligonucleotides will go into solution immediately upon addition of water or buffer. At higher stock concentrations (greater than 4 micrograms per microliter) or if the sample is difficult to dissolve, it may be necessary to heat for five minutes at 55 degrees C before vortex mixing. Before using, microfuge the solution and determine the A260 of the supernatant. This should give an indication of whether the oligonucleotide has completely dissolved.

8. It looks as if there is something insoluble at the bottom of the sample tube. What is it?

Occasionally there may be some carryover of solid support to the sample tube. It appears as an insoluble, granular solid. It is chemically inert and will not affect enzyme reactions. The solid can be removed by microfuging and transferring the oligonucleotide solution to a new, sterile tube.

9. Are the oligonucleotides purified?

All of the oligonucleotides synthesized by Oligos Etc. undergo a primer grade purification. This is a proprietary extraction and precipitation protocol specifically to remove excess salts and organics. The resulting counter ion is ammonium. The oligonucleotides are suitable for most PCR work. Primer grade purification is provided for all oligonucleotides free of charge. Other levels of purification such as gel or HPLC purification are available and may be requested at an additional charge.

10. Do synthetic oligonucleotides have phosphate groups (PO4) or hydroxyl groups (OH) at the 3' and 5' termini?

Synthetic oligonucleotides have terminal hydroxyl groups. However, you may request specific modifications of your oligonucleotide to include 5', 3', or both 5' and 3' phosphate.

(There is a charge for the modifications.)

11. What does "Loop Tm" mean on the computer analysis sheet?

Loop Tm is the temperature below which your oligonucleotide will develop secondary structure. To prevent secondary structures from forming, work above the Tm. (If the computer analysis sheet that came with your oligonucleotide reads 'no stable secondary structure', then the above does not apply to that oligonucleotide. In this case, the oligo should not fold onto itself.) All melting temperatures provided are calculated at 1 molar salt concentrations.

12. What is the optimal annealing temperature for my oligonucleotide pair?

The optimal annealing temperature (OAT) is the temperature where PCR of a given fragment is successful. This temperature varies depending on the primers and the melting temperature of the entire fragment. Empirically OAT is normally between 5 and 10 degrees Centigrade lower than the primer to target Tm of the lowest Tm primer in a pair. This empirical observation gives a good starting annealing temperature (T A ) for amplification reactions.

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