FAQ - DNA Oligo Synthesis for qPCR, PCR and NGS

FAQ - Custom Oligonucleotides

General & Ordering

• What documentation do I receive with my oligos?

  Each oligo is delivered with a Data Sheet and a QC Report.

  The Data Sheet includes sequence information, amount supplied, and recommended resuspension volume; the QC Report summarizes the analytical data (e.g. ESI-MS, and HPLC if ordered).

• What is the shortest and longest sequence I can order?

  Standard DNA oligos are available from 6 bases up to 100 bases in length.

  Longer sequences may be possible on request. For feasibility assessment and a customized quotation, please contact techsupport@invitek.com.

• Which types of oligos can I order (DNA, modified)?

  We offer a broad range of DNA oligonucleotides, with multiple modification and purification options suitable for PCR, qPCR, cloning, NGS library prep and other applications.

  If you have a complex design (e.g. heavily modified antisense oligos, multiplexed probe panels), our technical team can help evaluate feasibility.

• What modifications are available?

  We support a wide portfolio of modifications including (but not limited to):

  • Fluorescent dyes (e.g. 5’ reporter dyes for qPCR)
  • Quenchers (3’ or internal)
  • Attachment groups (biotin, amino, thiol, etc.)
  • Spacers and linkers
  • Backbone modifications and analogs (e.g. phosphorothioate)
  • Intercalating groups and antisense-relevant modifications

  Standard options are listed on our online ordering page.

  If you do not see the modification you need, there is still a good chance we can work with it, especially if it is commercially available as a CPG, phosphoramidite, or NHS ester. Please contact techsupport@invitek.com for feasibility and quotation.

• What purification methods are available and what purities do you guarantee?

  We offer the following purification options:

  • Desalt
  • No formal purity guarantee
  • Suitable for many standard PCR and screening applications
  • Cartridge purification
  • No formal purity guarantee; typical purity ~65–80%
  • Good balance of cost and purity for many routine uses
  • RP-HPLC (Reverse-Phase High Performance Liquid Chromatography)
  • Guaranteed purity ≥85%
  • Recommended for sensitive applications (qPCR probes, cloning, NGS, etc.)

  If you are unsure which purification level you need, our technical team can advise based on your application.

• Do you offer oligo design assistance?

  Yes. You can:

  • Use our online primer/probe design tools, and/or
  • Request free consultative design support from our technical services scientists via the online contact form or by emailing techsupport@invitek.com.

  We can help with primer/probe design, multiplexing strategies, dye/quencher combinations, and basic in silico quality checks.

• When designing my sequence, where should I place modifications?

  Placement depends on your application:

  • qPCR probes (e.g. TaqMan®-style): typically a 5’ reporter dye and a 3’ quencher work very well.
  • Hybridization probes or FRET pairs: may require internal labels specific dye/quencher spacing.
  • Capture oligos / immobilization: an amino, thiol or biotin is often placed at the 5’ or 3’ end, depending on surface chemistry.

  Because optimal placement can vary by assay, we recommend contacting techsupport@invitek.com for guidance if you are unsure.

• How is the melting temperature (Tm) of an oligo calculated?

  We use different approaches depending on oligo length:

  • For sequences ≥ 15 bases, we use a nearest-neighbor method (thermodynamic model that considers base stacking interactions).
  • For sequences ≤ 14 bases, we use a simplified formula (a modification of the Marmur-Doty equation).

  Keep in mind that Tm predictions are estimates; actual Tm can be influenced by buffer composition, salt concentration, and the presence of additives.

• How should I choose the annealing temperature (Ta) for PCR?

  The annealing temperature depends on primer Tm, primer composition, and target sequence. As a general starting point:

  • Set Ta approximately 5 °C below the primer Tm.

  A more detailed formula for the optimal annealing temperature (Ta_opt) is:

  Ta_opt = 0.3 × (Tm of primer) + 0.7 × (Tm of product) – 14.9

  Where:

  • Tm of primer is the melting temperature of the less stable primer-template pair
  • Tm of product is the melting temperature of the PCR product

  If Ta is too high, primers may not anneal efficiently, also reducing yield.

  We recommend performing a small gradient PCR to empirically determine the optimal Ta for your assay.

• How are oligonucleotides synthesized?

  Our oligos are produced by phosphoramidite solid-phase synthesis:

  • 1. The first base is attached to a solid support.
  • 2. Each subsequent base is coupled in a stepwise manner.
  • 3. After synthesis, the oligo is cleaved from the support, deprotected, purified (as ordered), and finally dried.

  This well-established chemistry ensures high reproducibility and compatibility with a wide range of modifications.

• How do you determine the quantity of oligo I receive?

  We determine the amount by:

  • 1. Measuring the absorbance at 260 nm (A260) using a UV-Vis spectrophotometer.
  • 2. Converting absorbance to concentration using the Beer-Lambert law and the calculated extinction coefficient.
  • 3. Reporting the result in customary units such as nmol and optical density (OD).

  The exact amount shipped is printed on the tube label and on the Data Sheet.

• If I order a 0.2 µmol synthesis scale, will I receive 0.2 µmol of oligo?

  No. The synthesis scale refers to the starting amount of the first base on the solid support, not the final yield.

  At each coupling step the efficiency is <100%, and="" subsequent="">cleavage, deprotection and purification also reduce yield.

  If you need a specific final amount (rather than ordering by synthesis scale), please let us know; in many cases we can target a requested quantity or advise on the appropriate scale.

• What quality control (QC) methods do you use?
  • ESI-MS (Electrospray Ionization Mass Spectrometry):

  Used as a gold standard method to confirm oligo identity. Provided free of charge for appropriate products.

  • Analytical HPLC:

  Available on request for an additional fee to assess purity and verify the purification profile.

  If you have project-specific QC needs (e.g. additional documentation, certificates), please contact techsupport@invitek.com.

• Can I obtain your ISO 9001 / ISO 13485 certificates?

  Yes. If you require copies of our current certificates for your quality system or supplier qualification files, please email techsupport@invitek.com.

• What are the recommended storage conditions for my oligos?
  • Room temperature (dry or in TE): typically stable for 3–6 months.
  • 4 °C (short-term storage): stable for approximately 1 year.
  • –20 °C (long-term storage): stable for approximately 2 years.

  Oligos may be stored dry or in solution solution (e.g. water or TE buffer) without significant differences in stability, provided they are kept clean and tightly sealed. Avoid repeated freeze–thaw cycles; we recommend preparing aliquots for frequent use.

• I do not see any material in the tube / plate well. Is my oligo actually there?

  Yes – this is common. Oligos are shipped dry and often appear as a nearly invisible thin film or clear glaze on the bottom or wall of the vessel.

  We recommend:

  • 1. Briefly the tube or plate before opening, to collect any material that may have moved during shipping.
  • 2. Resuspending in the recommended volume of water or buffer.
  • 3. Confirming presence with an A260 measurement if desired.

• How do I resuspend my oligos?

  We recommend:

  • 1. Prepare molecular biology grade water or TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).
  • 2. Add the volume indicated on your Data Sheet to achieve the desired stock concentration (e.g. 100 µM).
  • 3. Vortex gently or flick the tube and briefly centrifuge to collect the solution.
  • 4. Allow the oligo to fully dissolve (a few minutes at room temperature is usually sufficient).

  We do not recommend DEPC-treated water for resuspension or storage, as it may be slightly acidic and can cause depurination and degradation of the oligo over time.

  A common workflow is to prepare a concentrated freezer stock (e.g. 100 µM), then make diluted working aliquots as needed for short-term use.

• How do I prepare a 100 µM stock solution from my oligo?

  A convenient shortcut:

  • Take the number of nmol indicated on the tube label / Data Sheet.
  • Multiply by 10to obtain the volume in µL needed for a 100 µM solution.

  Example:

  • If the label says 5 nmol, add 50 µL to obtain a 100 µM stock.

  Note: The recommended resuspension volume is also indicated on the technical Data Sheet, so you don’t need to calculate it yourself unless you want to use a different concentration.

• I need a modification or format that isn’t listed online. Can you still make it?

  Often, yes. If the required building block (CPG, phosphoramidite, NHS ester, etc.) is commercially available and compatible with our synthesis platform, we can frequently incorporate it.

  We also support special formats (e.g. plates, mixed or pooled oligos) for certain projects. Please send your requirements to techsupport@invitek.com. for evaluation.

• Who should I contact if I have technical questions or need help choosing the right oligo?

  Often, yes. If the required building block (CPG, phosphoramidite, NHS ester, etc.) is commercially available and compatible with our synthesis platform, we can frequently incorporOur technical support team is happy to help with:

  • Assay design and optimization
  • Choice of modifications, dyes and quenchers
  • Purification level and scale selection
  • Troubleshooting performance issues

  You can reach us via the online contact form or directly at techsupport@invitek.com.