Oligos: Frequently Asked Questions (FAQs)

 

How does Sigma quantitate oligos?

We carefully measure the O.D. value of custom oligos using an UV spectrometer.  Additional information regarding the equations used to determine the O.D. value is available: Quantitating DNA.

 

I ordered a 0.2 µmole scale oligo. Does this mean that I will receive 0.2 µmole of product?

The scale of synthesis represents the µmole of starting material for the first base of synthesis.  The overall µmole yield of full-length oligonucleotide depends on the average coupling efficiency of each base and on the number of couplings (length of oligo).  Sigma quotes a coupling efficiency of 98-99%.  Yields of full-length oligo can vary considerably based on length and type of purification requested.  Sigma has set standard minimum yield guarantees based on the synthesis and purification of your oligo.  The minimum guaranteed yields are based on absorbance at 260 nm (optical density units or O.D.s) rather than µmole amounts.

 

What oligonucleotide modifications does Sigma offer?

Sigma offers over 200 modifications for both DNA and RNA oligonucleotides and probes. Sigma can attach fluorescent dyes to oligos at the 5'-end, 3'-end and internal positions. Our product offering includes common dye modifiers such as 6-FAM™, HEX™, and TET™, as well as exotic modifications.  View our offering at: sigma.com/mods.

In addition to standard fluorescent modifications, we have the capabilities to provide dual-labeled probes including Molecular Beacons, LightCycler® Probes, and Scorpions® Probes.

 

I received my oligonucleotide but there seems to be nothing in the vial. Where is the oligonucleotide?

All oligos are provided dry unless otherwise specified.  It may be difficult to see the oligo in the bottom of the vial.  You can be assured that the oligo is in the vial with the quantitative information located on the vial label.  We recommend that you quick spin each vial prior to opening.

 

What purification methods are available?

Several types of purification are available (see also Oligonucleotide Purification):

  1. Desalting: Every oligo is desalted to remove residual by-products from synthesis deprotection and cleavage.
  2. RP1 (Reverse-Phase Cartridge): Separation on a reverse-phase cartridge offers the next level of purity.
  3. HPLC: Efficient purification method for oligos with fluorophores and large scale synthesis.
  4. Poly-Acrylamide Gel Electrophoresis (PAGE): Recommended when a highly purified product is required.

 

When designing my oligo, should I locate modifications at any specific site?

We recommend 5' modifications when possible; however 3' and internal modifications are also available.

 

What are the recommended storage conditions for oligonucleotides?

We recommend you store oligonucleotides at 4°C for short term use (1-2 weeks).  For intermittent use, store at –20°C.  An important consideration in oligo stability is to prepare stock solutions with nuclease-free sterile water or 1x TE buffer.  Fluorescently labeled oligos should be stored in light-free conditions.  For additional information, see Handling Guidelines for DNA and RNA Oligonucleotides.

 

How does Sigma calculate the melting temperature (Tm) of oligonucleotides?

The melting temperature (Tm) is calculated using the nearest-neighbor method.  The factors that affect the Tm of an oligo in an application include: salt concentration, strand concentration, the presence of denaturants as well as the sequence and the length of the particular oligonucleotide.  Sigma uses standard PCR conditions to determine the Tm (50 mM NaCl and 0.5 µM oligonucleotide). Click for more information.

 

Will Sigma anneal two DNA oligonucleotides?

We do provide annealing services for DNA oligos.  Please email dnaoligos@sial.com.

You may also view our protocol for annealing oligos.

 

Does my oligonucleotide have a phosphate on the 5' or the 3' end?

All of our custom oligonucleotides are synthesized with a hydroxyl group on both the 3’ and 5’ ends.  However, if requested, we can synthesize your oligo with a 5’ and/or 3’ phosphate.

 

What is the longest oligonucleotide that Sigma will synthesize?

Sigma can synthesize oligos up to 120 bases in length. Please note that yield and quality can be affected by base composition. Oligos greater than 110 bases can only be synthesized on the 0.2 or 1.0 µmole scale with PAGE purification. For oligos greater than 120 bases, please contact your local Sigma office for pricing and feasibility or visit: sigma.com/longoligos.

 

If I left my oligos on the lab bench over the weekend, will they still work?

Dry oligonucleotides have tremendous stability.  Oligos in solution have a more finite shelf-life.  For most situations, resuspended oligos should still work well, even if left at room temperature for a couple of days.  More information: Handling Guidelines for DNA and RNA Oligonucleotides.

 

Does Sigma offer oligonucleotide design assistance?

Sigma is pleased to offer OligoArchitect ™ for all of your primer and probe design requirements. OligoArchitect is complimentary and includes both our online design tool and our consultative service.

Try our online design and oligo calculator tools.

 

How do I make a 100 µMolar stock solution of my oligo?

Sigma provides quantitative data on your oligo using nmoles, optical density units (O.D.) and micrograms.  This allows simple preparation of stock solutions for whatever units you prefer.  A general rule states that for any oligo, the number of nmoles x 10 will give you the amount of solvent to add in microliters for a 100 µM stock solution.  100 µM is also equal to 100 pmol/microliter for those who wish to work with pmole amounts.  For additional information on resuspending and handling oligos: Handling Guidelines for DNA and RNA Oligonucleotides.

 

 

Materials