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How does Sigma-Aldrich quantitate their oligos?
We carefully measure the O.D. value for your custom oligonucleotide using an UV spectrometer. To inspect the equations used to determine the O.D. value, view our Quantitating DNA web page for more detailed information. If you have any questions regarding the yield of your oligo, then please contact our technical support staff.
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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 (or the length of the oligo). Sigma-Aldrich quotes a coupling efficiency of 98-99%. Yields of full-length oligo can vary considerably based on length and type of purification requested. Aldrich has set standard minimum yield guarantees following 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. If we exceed these minimum guarantees, then we ship the full amount obtained from the synthesis.
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What oligonucleotide modifications does Sigma-Aldrich offer?
Sigma-Aldrich offers numerous oligonucleotide modifications including biotin, terminal amines, terminal phosphates, fluorescein, 6-FAM, HEX and TET just to name a few. However, if you have a specific modification requirement, please call your account representative for the availability of the modification.
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I received my oligonucleotide order, but there is nothing in the vial. Where is the oligonucleotide?
All of our custom oligonucleotides are provided dry unless otherwise specified. It may be difficult to see the oligo in the bottom of the vial. However, be assured that your oligo is in the vial with the quantitative information located on the vial label.
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What types of oligonucleotide purification does Sigma-Aldrich offer?
Several types of purification are available (see also Oligonucleotide Purification):
- Desalting. Every oligo is desalted free of charge to remove residual by-products from synthesis deprotection and cleavage.
- RP1 (reverse-phase cartridge). Cartridge purification provides 80-90% pure full-length product.
- HPLC. Provides 90-97% full-length product and is the method of choice for purifying larger quantities of oligos (>1 mmol scale of synthesis).
- Poly-Acylamide Gel Electrophoresis (PAGE). Provides 95-99% full length product purity. The preferred method for oligos longer than 45 bases.
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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.
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What are the recommended storage conditions for oligonucleotides?
We recommend that you store oligonucleotides at 4ºC 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. Alkaline phosphatase conjugated oligos should be stored at 4°C. For more information, see You and Your Oligo.
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How does Sigma-Aldrich calculate the melting temperature (Tm) of my oligonucleotide?
We calculate the Tm using the nearest-neighbor method. The factors that affect the Tm of an oligo in an application are: salt concentration, strand concentration, the presence of denaturants as well as the sequence and the length of the particular oligonucleotide. Sigma-Aldrich uses typical PCR conditions to determine the Tm (50 mM NaCl and 0.5 uM oligonucleotide). Check our Melting Temperature tech note for more information.
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If I order two oligonucleotides, will Sigma-Aldrich anneal them for me?
We do not offer oligonucleotide-annealing services. However, view our DNA Annealing page for a standard annealing protocol.
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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 the 5' ends. However, if requested, we can synthesize your oligo with a 5' and/or 3' phosphate.
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What is the longest oligonucleotide that Sigma-Aldrich will synthesize?
We can synthesize oligos as long as a 130-mer. Please note that yield and quality can be affected by base composition. Oligos greater than 110 bases can only be synthesized at the 1.0 umole scale.
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How do I place an order for Sigma-Aldrich products?
There are several ways in which you may place an order.
- You can direct your order to us by fax at (281) 363-2212. Order forms are available through the Fax-Forms web page; for long oligos; for multiple oligos.
- Orders may be sent by email to gorderentry@sial.com. Instructions for sending emails can be found on the Ordering Information web page.
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Does Sigma-Aldrich make RNA oligonucleotides?
2'-O-Me derivatives of RNA are very common and quite straight forward to synthesize. We do not synthesize native RNA due to the post-synthesis stability issues. Please consult our technical support group for more information.
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If I left my oligos on the lab bench over the weekend, will they still work?
Dry oligonucleotides have a tremendous stability. Oligos in solution have a more finite shelf-life. Anyone can forget to return their primers to the freezer. However, the answer to this question lies with the quality of solution used to resuspend the oligos. For most situations, resuspended oligos should still work well, even if left at room temperature for a couple of days.
Will Sigma-Aldrich help design my oligonucleotide?
Our technical staff would be happy to answer any questions you may have about primer design. Additionally, you may determine the melting temperature, molecular weight, GC content, significant structure and primer dimer by using the online calculations feature. If you have additional questions, please contact our technical support group.
How do I make a 100 µMolar stock solution of my oligo?
Sigma-Aldrich provides quantitative data on your oligo using nanomoles, optical density units and micrograms, so stock solutions may be made using whichever units you prefer. However, a handy rule of thumb states that for any oligo the number of nanomoles 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 further information on resuspending and handling oligos, please see our "You and Your Oligo" tech sheet.
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