Summary
Annealing protocols are fairly standard and can be found on the internet through search engines or on different company websites. The protocols are generally the same but may differ in reagents and incubation times / temperatures. Eurofins has a sample annealing protocol attached to this FAQ. The protocol may be modified or optimized to your specifications.
NOTE: Annealed dsDNA oligos should be stored at –20 °C or kept on ice when on the benchtop.
For your convenience Eurofins has an added feature when ordering through the website to have the reverse complement sequence.
Annealing Buffer: 10 mM Tris, pH 7.5–8.0, containing 50 mM NaCl and 1 mM EDTA
1x TE Buffer: 10 mM Tris, pH 7.5–8.0 containing 1mM EDTA
Step 1. Resuspending the Oligonucleotides: Resuspend both complementary oligonucleotides at the same molar concentration, using Annealing Buffer (see note below). For convenience, keep Annealing Buffer volume below 500 μL for each oligo. Annealing should perform well over a wide range of oligo concentrations. For larger scale oligo syntheses, it may be necessary to use larger volumes that can be aliquoted after resuspension.
Step 2. Annealing the Oligonucleotides: Mix equal volumes of both complementary oligos (at equimolar concentration) in a 1.5-mL microfuge tube. Place tube in a standard heatblock at 90–95 °C. Remove the heatblock from the apparatus and allow to cool to room temperature (or at least below 30 °C) on the workbench. Slow cooling to room temperature should take 45–60 minutes. Store on ice or at 4 °C until ready to use. An alternative procedure for annealing involves the use of a thermal cycler. Dispense 100 μL aliquots of the mixed oligos into PCR tubes (500-μL size). Do not overlay the samples with oil. Place the tubes in a thermal cycler and set up a program to perform the following profile: (i) heat to 95 °C and remain at 95 °C for 2 minutes, (ii) ramp cool to 25 °C over a period of 45 minutes, (iii) proceed to a storage temperature of 4 °C. Briefly spin the tubes in a microfuge to draw all moisture from the lid. Pool samples into a larger tube, store on ice or at 4 °C until ready to use.
Step 3. Long Term Storage: It may be necessary to aliquot and lyophilize the annealed sample. After drying, the sample may be stored at –20 °C in a desiccated container. Resuspend the annealed oligos at the desired concentration with sterile distilled water. The annealed pair of oligonucleotides is ready for use.
NOTE: Oligos may also be resuspended in either 1x Ligase Buffer or 1x Kinase Buffer instead of the above Annealing Buffer (prior to annealing).
Unmodified oligos can be used for at least 12 months after purchase when stored wet at –20 °C. For long-term storage, oligos should be stored dry at –20 °C. If numerous experiments are planned using the same sequence, aliquot the sample, dry all aliquots, and store at –20 °C. If the oligos are stored wet, avoid repeated freeze-thaw cycles as this process can lead to physical degradation of the oligo. Oligos generally last longer in TE than in water. Careful handling is recommended to avoid the possibility contamination with nucleases or bacteria.
Before opening, spin the tube at 1000 rpm for 5 min to ensure that the oligonucleotides are at the bottom of the tube. Oligonucleotides should be resuspended in a sterile buffered solution (e.g., TE at pH 7.0). Vortex oligonucleotides thoroughly after resuspension. The oligonucleotides may not readily dissolve in sterile, distilled water. The addition of NaOH to the water until the pH rises to 7.0 may help. If the oligonucleotides are resuspended at pH <7.0 (deionized water may have a pH as low as 5.0), the oligonucleotide could begin to degrade and may lose functionality within a couple of weeks.
For optimal long-term storage, oligonucleotides should be stored dry at –20 °C in the dark. If numerous experiments are planned using the same oligonucleotide, prepare aliquots, dry them and store the aliquots at –20 °C. Use clean, sterile labware for all transfers.
Dual-labeled probes (fluorescence quencher or FQ probes) have a fluorescent reporter and a quencher at their 5′ and 3′ ends, respectively. These probes can be used in quantitative PCR systems that take advantage of the 5′→3′ exonuclease activity of Taq DNA polymerase. A probe specific for the sequence of interest is used together with specific PCR primers. This probe is designed to anneal between the PCR primers. During the extension phase of PCR, the 5′→3′ exonuclease activity of Taq DNA polymerase cleaves the fluorescent reporter from the probe. The amount of free reporter accumulates as the number of PCR cycles increases. The fluorescent signal from the free reporter is measured in real-time and allows quantification of the amount of target sequence. There are several design considerations to keep in mind when designing dual-labeled probes. Placement of the probe is important; the probe should be designed first, followed by the design of the PCR primers. The probe should anneal near the center of the amplicon, and the amplicon should be 50 to 150 bases long. The probe's melting temperature should be 68 °C to 70 °C. The probe should be at least 20 bases long to prevent nonspecific annealing. Finally, avoid designing the probe with a dG at the 5' terminus as dG is a weak quencher. Any dG in the primer should be at least 2 residues away from the 5' terminus.
Probe:
G-C Content: 30–80%
polyNNN: Avoid stretches of same nucleotide, especially 4 or more Gs
End Composition: No Gs at 5' end
Tm: 68–70 °C
Strand: Select strand with more Cs than Gs. If the compliment is used, make sure there are no Cs at the 3' end.
Length: Less than 30 bp
Design: Quencher on 3' end dye on 5' end
Primer:
G-C Content: 30-80%
polyNNN: Avoid stretches of same nucleotide, especially 4 or more Gs
End Composition: 5 bases at 3' end should have no more than 2 G/Cs. Try to use As at 3' end so primer dimers will be degraded more efficiently.
Tm: 58–60 °C
Product: 50 to 150 bases
Design: Choose after probe. Design as close to probe as possible without overlap. Have one primer cross one exon junction to amplify mRNA.