19 Primer resuspension

Primer resuspension and dilution

After receiving an envelope containing their order of oligonucleotides,  what should you do with these oligos?

Centrifugation and resuspension. Most oligonucleotides synthesized and shipped are delivered dried down and often appear as a white flakey substance in the bottom of the tube. Dried DNA is quite stable and, typically, is easy to resuspend in an aqueous solution. The first step is to briefly centrifuge the tubes before opening them. This ensures that any dried DNA that may have become dislodged during shipping is brought down to the bottom of the tube. For long-term storage, the oligos should be resuspended in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). Tris (tris(hydroxymethyl)aminomethane) acts as a buffer, helping to maintain a constant pH for the solution. EDTA (ethylenediaminetetraacetic acid) prevents nuclease digestion of the DNA.

Alternatively, nuclease-free water, pH 7.0, can be used for resuspending oligonucleotides, but it will not modulate pH over time as will TE buffer. Use ofmolecular biology–grade water is preferable, as water from a deionizing system (such as Millipore) can be acidic, with a pH as low as 5.0.

Concentration and dilution.  The concentration at which your oligos should be resuspended is largely up to the discretion of the researcher, and is dependent on how the oligo will be used in future experiments. Oligonucleotides are stable when stored over a large range of concentrations; however, for optimal stability we recommend concentrations no less than 1 µM, and no greater than 10 mM. Resuspension calculations can be made using the yield information that is provided both on the oligo tubes themselves and on the oligo product specification sheets provided with the order. For your convenience and utility, this yield information is presented in optical density (OD) units, mass (mg), and copy number (nmol).

Our standard recommendation is to resuspend oligos to a 100 µM stock concentration, as this is a simple concentration to make and is highly versatile for subsequent dilution purposes

Storage

Optimal conditions for standard DNA oligonucleotides.  The most stable temperature at which to store a standard DNA oligonucleotide is -20 °C. An oligo stored at –20 °C is stable for at least 24 months when either dried down, or resuspended in TE buffer or nuclease-free water. Standard DNA oligos dried down or stored at 5°C in TE buffer or water were found to be stable for long periods; however, better stability is achieved by freezing if the oligos are to be stored for extended periods. Standard DNA oligos stored at 37 °C are stable for at least 6 weeks in water, or 25 weeks dried down or in TE. Storage at higher temperatures is not recommended; however, accidents can happen, such as oligos being left on the bench top overnight or freezers failing at the weekend, and it is important to understand that the stability of the oligo should not be affected. Regardless of the storage temperature, TE buffer, rather than water or dried down, is optimal for long-term storage.

Follow these steps for primer resuspension calculations

1. Find the # of nmoles of primer in your tube: 

2. Convert this number into umoles since the concentration you want is 100 uM:

3. If you want a primer solution that is 100 uM, you need to figure out the volume that will get you to this concentration

Your calculated umoles of primer =           Desired molarity = umoles

Volume you will resuspend in                                                         liter

Be prepared to solve these on your own, no matter what primer quantity or desired concentration that I give you.

• Once you have your 100 uM primer tubes, you can combine F + R primers into a primer mix that has both primers at a concentration of 10 uM (e.g., figure out how to get 10 uM of one primer. You’ll add an equal volume of the other primer so that BOTH are at 10 uM).
• Do a CV=CV equation for each primer independently, but remember to subtract both primer volumes when calculating the water volume you will use.
• This 10 uM dilution containing F and R primers is the tube you will use for your PCRs.