> DNA (Nucleic Acid) Synthesis Services


The Macromolecular Synthesis group offers several nucleic acid synthesis services. Custom oligonucleotide synthesis is available at several scales. Other services include special chemistry oligo synthesis, RNA synthesis, oligo purification, and an inventory of stock sequencing primers available for immediate purchase and pick up.

Available Stock Primers:

M13(-21) 5;-TGTAAAACGACGGCCAGT-3'
M13 Reverse 5'-CAGGAAACAGCTATGACC-3'
T7 5'-TAATACGACTCACTATAGGG-3'
T3 5'-ATTAACCCTCACTAAAGGGA-3'
SP6 5'-ATTTAGGTGACACTATAG-3'

How to Order

Select Scale

Several scales of synthesis are available. The scale chosen depends on the amount of oligo required. For many methods such as priming PCR and sequencing reactions, a very small amount of oligonucleotide is needed. In general, it is advisable to order the smallest scale synthesis possible.

The theoretical yields per base from the various scales are listed in a table below:

Scale Theoretical Yield/ Base
40 nmol 0.25 — 0.5
200 nmol 1 — 1.25
1 µmol 5 - 6
10 µmol 40

A number of factors including oligo length and sequence can result in actual yields that are significantly lower than the theoretical yields listed above.

Select a delivery option for your oligonucleotide.

The fastest option is unpurified product in ammonium hydroxide solution. Orders delivered this way can be released significantly faster than any other option, and an aliquot of several micro liters can be dried down for quantification and use in just a few minutes in your lab. The Hartwell Center can also lyophilize your order before delivery. Drying an oligonucleotide adds a significant amount of time, so it may be available later than an oligo ordered in ammonium hydroxide. Purification of oligonucleotides is not routinely available. The Hartwell Center's synthesizers feature high coupling efficiencies, and the few failure sequences that are formed are capped to eliminate products with internal deletions. Extensive testing by the Hartwell Center staff indicates that purification is unnecessary for primer-length oligonucleotides in the most common applications. The Hartwell Center recommends purification only for particularly sensitive experimental techniques and unusually long oligos. If you have a question about the recommended purity for a particular application, please contact the Macromolecular Synthesis Group.

Enter the label and sequence for your order.

The label is the name you give to the sequence. When entering the sequence, use the standard characters listed below. Inosine, modified bases, and mixed bases are not allowed in the 3´ terminal base. If you require a modified base in the 3’ position, it may be possible to complete your order as requested. Please contact the synthesis staff for synthesis options.

A G C T I

M = (A or C)
W = (A or T)
Y = (C or T)
V = (A or C or G)
R = (A or G)
S = (C or G)
K = (G or T)
H = (A or C or T)
D = (A or G or T)
N = (A or G or C or T)
B = (C or G or T)
T = (T or U)

Ordering RNA

RNA Orders can be placed using the Hartwell Center’s Online Ordering system. Both strands of a duplex must be ordered. When ordering select “RNA” and “0.2 umol” from the appropriate drop-down menus. When entering the sequences, substitute T for U. If you require dT at the 3’ terminal bases, please specify this in the comment box on the order form. RNA will be synthesized and completely deprotected by the Hartwell Center staff. In addition, the oligoribonucleotides will be desalted, quantified, and lyophylized. Quality control testing will be done on every oligoribonucleotide using MALDI-TOF mass spectrometry. The investigator will be responsible for annealing and subsequent purification of duplex RNA, if desired. Due to the complex and labor-intensive nature of RNA synthesis, turnaround time will be significantly longer than that of DNA synthesis.

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Quality Assurance

In order to ensure the highest quality syntheses possible, the Hartwell Center staff have established maintenance and quality control policies. All of the synthesizers and accessory equipment receive routine maintenance from the Hartwell Center staff. The synthesizers are all under service contracts that include annual preventive maintenance from the manufacturer’s service engineers.

All oligonuclueotides are quantified by UV absorbance A260 readings. Oligos with low yields are flagged for additional quality control or resynthesis.

The Hartwell Center routinely runs quality control tests on synthetic oligonucleotides. Oligonucleotide longer than 70 bases and special chemistry syntheses are automatically flagged for quality control. In addition samples are taken from routine synthesizer runs and tested to evaluate the quality of the synthesis and the proper operation of the synthesizers. Investigators can request quality control of any order. This will delay delivery by as long as a few days.

Two quality control methods are currently used to monitor oligonucleotide syntheses. Oligonucleotides can be evaluated by Polyacrylamide Gel Electrophoresis (PAGE). Oligonucleotides are electrophoresed between confirmed markers of a similar size. PAGE quality control is useful for oligonucleotides in the size range of 20 bases up to the longest oligonucleotides synthesized.

Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) mass spectrometry is also used to evaluate olignucleotides up to 70 bases in length, see proteomics.

The synthetic chemistry used by the Hartwell Center’s synthesizers is very reliable, and incorrect sequences and deletions are rare. If your experiment does not work as expected, please investigate reagents and protocols. If after troubleshooting you suspect the oligonucleotide is responsible, contact the Macromolecular Synthesis group at x2558. We will advise you on an appropriate aliquot of the oligonucleotide to submit for testing.

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Instrumentation

Oligos are synthesized on one of two ABI 3900 DNA Synthesizers. Each ABI 3900 is capable of 48 parallel syntheses. All oligos are quantified by A260 absorbance on a Molecular Devices Spectramax Plus 384. This UV/visible spectrophotometer plate reader is also used for quality control of dye-labeled oligos and other applications. High capacity Savant SpeedVac systems and other standard laboratory equipment are also available.

 

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Fees

Please follow this link to a general fees page.

Related Methods

Storage and Handling of Synthetic Oligonucleotides

Recommended storage for most oligonucleotides that have been lyophilized or are in ammonium hydroxide is at —20°C. When stored this way, DNA oligonucleotides are stable almost indefinitely. RNA oligonucleotides stored as above are stable for approximately 2 months.

Olignucleotides should be resuspended in dH2O, aliquoted, and frozen. Aliquots should then be thawed as needed. Working aliquots may be stored at 4°C for up to one month. Avoid repeated freeze thaw cycles. Repeated freezing and thawing can cause degradation and poor experimental results.

Synthetic RNA should be stored at –80 C. RNA should only be resuspended in RNAse free buffer and aliquoted to RNAse free tubes. As with synthetic DNA, RNA should not be repeatedly frozen and thawed. Even with good handling, RNA is much more labile than DNA and will have a limited storage life.

Quantification of oligonucleotides by UV absorption

Oligonucleotides are most accurately and conveniently quantified by the measured absorbance of UV light of the sample in a spectrophotometer. For best results when performing UV absorbance analysis, use clean quartz cuvettes and blank each sample with the same solution used to dissolved the sample.

An optical density (O.D.) unit is defined as the amount of a substance dissolved in 1.0 ml that will give an absorbance reading of 1.00 in a spectrophotometer with a 1 cm path length. Because the average absorbance maximum of the four bases is approximately 260 nm, optical density for DNA is commonly reported for this wavelength. The formula OD can be calculated as follows:
Formula OD = A260 * stock volume in ml * dilution factor

Dilution factor = final dilution volume in ml / aliquot volume in ml

Ethanol Precipitation of Oligonucleotides

Oligonucleotides which have been synthesized by the Hartwell Center with cartridge purification have had synthesis by-products removed, have been desalted and can be used for all applications without further processing. Oligonucleotides which have been synthesized without cartridge purification can be isolated from minor impurities (such as protecting group by-products benzamide and isobutyrylamide, short failure sequences, salts and small amounts of solvents) by precipitation from alcohol and water solutions using the following method. Precipitation also affords an easy opportunity to change the cation associated with the oligonucleotide. For example, the crude synthesis product is in the ammonium salt form, which may have limited solubility or inhibit some enzymes.

Precipitation Protocol:

  1. Dry the oligonucleotide (or a desired aliquot) using a vacuum centrifuge (such as a SpeedVac) without heating.
  2. Dissolve the dried oligonucleotide in 30 µL of dH2O and 5 µL of 3 M sodium acetate pH8.5 per ODU of oligonucleotide.
  3. Add 100 m µl/ODU of 100% ethanol, then vortex.
  4. Store at refrigerator (4°C) or freezer (-20°C) temperatures for about 30 minutes, then centrifuge in a microfuge at maximum speed for 5 minutes. For very short oligonucleotides (smaller than 15-mers), isopropanol may be substituted for ethanol to ensure complete precipitation.
  5. Remove the supernatant with a pipet or micropipet and discard, being careful not to disturb the pellet. Small quantities (less than several ODU or 100 mg) may not be visible.
  6. Again add 100 µl/ODU of 100% ethanol, mix briefly and centrifuge for 1-5 minutes.
  7. Remove the supernatant with a pipet or micropipet and discard, being careful not to disturb the pellet. Dry the oligonucleotide using a vacuum centrifuge without heating.
  8. Resuspend the oligonucleotide in dH2O and quantitate by absorbance.

Reference: "Evaluating and Isolating Synthetic Oligonucleotides", Applied Biosystems, Inc., 1987

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