DNA Synthesis

The DNA Synthesis Laboratory, located in Room 2360 of the University of Wisconsin Biotechnology Center, serves as a reagent source for molecular biology and genetics researchers. We provide oligonucleotides (oligos or primers) suitable for PCR, sequencing, site directed mutagenesis and other procedures. Available types, scales, and purity levels of oligos are listed below. The Synthesis Facility can also carry out many novel syntheses and oligonucleotide modifications, users need simply to contact the Biotech Center with their requests.

Biotechnology Center Contact Information	Phone (office): 608/263-9882 FAX: 608/265-6573
	E-mail: mjmorgan@wisc.edu

All data and reagents distributed by the University of Wisconsin Biotechnology Center DNA Synthesis facility are intended for research purposes only. They are not intended or certified for diagnostic or clinical use.

Custom Oligonucleotide Pricing and Scales

Below is a table showing pricing and other information for the types of oligonucleotide synthesis scales offered. To get specific information for a particular synthesis scale, click on the header for that scale. You will then be linked to ordering directions and information specific to that synthesis scale. Note that while only the 50 nmol "unpurified" PCR/sequencing oligos undergo routine quality control (QC) by Mass Spectrometry, this procedure can be carried out on any oligo the customer desires (including those not synthesized at this facility). The charge for this service is $5.00, and a hard copy of the data is provided. Also note that the 50 nmol (<60mer) purified synthesis scale at the Biotech Center requires an additional cartridge purification step, so there is a $5.00 charge for purification of these small scale oligos. In all other cases, an additional cost of $25 is assessed for cartridge purification and a base of $60 for HPLC. If you have questions please call the facility at 608-263-9882.

50 nmole (<60-mer)

All orders placed for this option will be cartridge purified as part of the cost, an additional $5.00 relative to the 50 nmole non-purified is added. If a customer orders a small scale oligo that is a 50-60mer, this is the selected synthesis choice by default. We have learned that these longer oligos are too "dirty" to use without first purifying them. The synthesizer used for this option is a PolyPlex II from Genomic Solutions. The instrument does NOT have a built in purification, so we at the Biotech Center manually do a RP cartridge purification on requested oligos. This is preferred for site-directed mutagenesis work and raises signal to noise ratio in numerous molecular biology applications. On the customer copy the total nanomoles/vial and micromoles are provided for each oligo. We lyophilize the product after elution because of the toxicity of ACN and the fact it will degrade the oligos over time. Turnaround time for this option is typically 24-36 hours, if the order is received before 3:00 P.M. Users are notified by telephone when their order is ready.

40 nmole (>60-mer or Modified)

All oligonucleotides longer than 60 bases and any small scale modified oligos (for a list of some of the available modifications and pricing information, click on Modifications ) will be made on an ABI 394 which does not include a purification step. If a modification is desired, note this in the sequence using a "Z" to designate the modified base, and make a second note of it in the "Comments" section of the oligo order form. Multiple modifications within a single oligonucleotide can be requested using W, X, or Y at the appropriate position within the sequence. The oligonucleotides synthesized in this option are analyzed by polyacrylamide gel electrophoresis (PAGE) to insure that laddering of the DNA (indicating synthesis failures) is minimal. In addition, the synthesis of these oligos is analyzed at each step by an integral trityl monitor in the synthesizer, so failures are usually detected right away. At a cost of $5 to the user the Biotech Center can run a Mass Spec Analysis of the DNA and provide a hard copy of the output. For more information about this, click here on Mass Spectrometry . These oligonucleotides can be cartridge purified for $25 (needs to be denoted in the "Comments" section) or HPLC purified for $50 (simply click on the button in the order form. Yield is decreased with purification. If you have any questions regarding purification simply click on Cartridge Purification. Turnaround time for this option is approximately 2-3 days, but can vary depending on modifier availability.

200 nmole (Standard or Modified)

Synthesis of oligonucleotides at this larger scale, either plain or modified (for a list of some of the available modifications and pricing information, click on Modifications ) will be made on an ABI 394 which does not include a purification step. If a modification is desired, note this in the sequence using a "Z" to designate the modified base, and make a second note of it in the comments section of the oligo order form. Multiple modifications within a single oligonucleotide can be requested using W, X, or Y at the appropriate position within the sequence. The oligonucleotides synthesized in this option are analyzed by polyacrylamide gel electrophoresis (PAGE) to insure that laddering of the DNA (indicating synthesis failures) is minimal. In addition, the synthesis of these oligos is analyzed at each step by an integral trityl monitor in the synthesizer, so failures are usually detected right away. At a cost of $5 to the user, the Biotech Center can run a Mass Spec Analysis of the DNA and provide a hard copy of the output. For more information about this, click here on Mass Spectrometry . These oligonucleotides can be cartridge purified for $25 or HPLC purified for a $50 base cost, but we need to know this before synthesis is initiated so indicate it by clicking the relevant request on the order form. Yield is decreased with purification. If you have any questions regarding purification simply click on Cartridge Purification. Turnaround time for this option is approximately 2-3 days, but can vary depending on modifier availability.

50nmole (Unpurified for PCR/Seq)

A newer higher throughput oligonucleotide synthesizer now on site at the UW DNA synthesis facility allows us to make smaller scale oligos at a greatly reduced price. These oligos are not purified at all, and are designed for use primarily in PCR and sequencing reactions. The Biotech Center uses these oligos regularly for in house sequencing and PCR projects. Unlike the previous instrument we at the Biotech Center will quantify ALL syntheses. Presently we are charging $0.25/base, which includes purification AND QC (there is NO set-up fee here). No modifications are available with this option, but unlike the previous instrument mixed bases ARE available. We are having routine success with 60-mers thus far, so the old length restrictions have been lifted (do note that any oligo over a 50-mer will automatically be purified, as we've found more success this way). Again, mass spec is done on all oligos at this scale, and there is no additional cost for this quality control analysis--it is built into the pricing. No hard copy of the results is routinely generated or provided to the user, but as with other synthesis options it is available for a small fee. Click here on Mass Spectrometry for more information about the utility of this analysis procedure. Ordering is carried out in a slightly different manner for this option.

1 micromol (Standard or Modified)

For very large quantities of DNA, the Biotech Center has available a 1.0 micromol scale synthesis for the users. Large-scale syntheses are available with the same assortment of modifications as the 40 and 200 nmole standard oligos, so when ordering use the same forms. If a modification is desired, note this in the sequence using a "Z" to designate the modified base, and make a second note of it in the comments section of the oligo order form. Multiple modifications within a single oligonucleotide can be requested using W, X, or Y at the appropriate position within the sequence. The oligonucleotides synthesized in this option are analyzed by polyacrylamide gel electrophoresis (PAGE) to insure that laddering of the DNA (indicating synthesis failures) is minimal. In addition, the synthesis of these oligos is analyzed at each step by an integral trityl monitor in the synthesizer, so failures are usually detected right away. At a cost of $5.00 to the user, the Biotech Center can run a Mass Spec Analysis of the DNA and provide a hard copy of the output. For more information about this, click here on Mass Spectrometry . These oligonucleotides can be cartridge purified for $25.00, but we need to know this before synthesis is initiated so indicate it in the "Comments" section of the order form. Yield is decreased with purification. If you have any questions regarding purification simply click on Cartridge Purification. Turnaround time for the 1.0 micromol option is approximately 2-3 days, but can vary depending on modifier availability.

Cartridge Purification

Cartridge purification of oligonucleotides is based on the differential hydrophobicity of tritylated (full length) vs. non-tritylated (failure syntheses) oligonucleotides (a reverse phase based approach). This procedure can be carried out at a cost of $5/oligonucleotide on our 50nmole scale and $25/oligo for 200 nmole or 1 micromole scales. The protocol we use is nearly as effective as HPLC purification at removing failure syntheses (read the HPLC Purification section for reference). It has been our experience here that cartridge purification provides highly enriched full length product (>90%) when performed on oligos of 50 or fewer bases in length. If cartridge purification is performed on longer primers, the eluted product is still predominantly composed of full length product, but a higher percentage of failure sequences end up in the eluted final product since the hydrophobicity differences between tritylated and untritylated oligos decreases as the oligo length increases. User feedback on the quality of the longer purified oligos has been very positive, but we wanted to bring the potential limitations to your attention to help in assessing your needs. Additionally, we have had no success in purifying any oligo larger than 90 bases in length. Also keep in mind that final yield will be decreased by purification. Oligonucleotides >60 nucleotides in length inevitably will have shorter products present, even after cartridge purifcation, as noted above. If it is absoloutely essential that you have pure full-length product of a longer oligonucleotide, you will have to purify it yourself by preparative PAGE or request HPLC purification. Most of our customers who are using very long oligos in, for example, cloning projects where they ultimately will be constructing a long stretch of cloned double stranded DNA, find they don't need to purify but can just sequence several clones containing the synthesized sequence in order to find their full length construct.

HPLC Purification

The DNA Synthesis Facility at the Biotech Center now has the ability to perform HPLC purifications upon user request. We utilize a reverse phase column that elutes DNA by both increasing length and hydrophobicity. In contrast to cartridge purification, this technique bypasses the need for the trityl group and the harsh reagents required to remove the trityl post-purification. We can separate the full length oligo from the N-1 peak up to a length of about 40 nucleotides using our standard solvent system. For longer oligos other solvent systems can be employed (such a request comes with a significant cost increase), though the system as presently configured still generates 95+% purity in longer oligos. Standard HPLC purification for such oligos will be $60/sample, regardless of whether purification is requested on 40nmole, 0.2 µmole or a1.0 µmole scale.
Singly labeled oligos (e.g., 5' FAM, HEX, TET, etc.) also utilize standard solvent systems since the label is hydrophobic and allows separation from unlabeled failure sequences. Cost for purification for these will also be $60/oligo. Multiply labeled oligos (e.g., those used in TAQMAN assays or other FRET applications, or labeled with multiple spacers, etc.) require more tweaking and analytical runs prior to the preparative run to insure collection of the correct final product. Pricing for these is subsequently higher, $85/oligo. As above, pricing is independent of scale.
In addition to the purified product, user will be provided with documentation including a digitally generated run log, and an analog run profile showing the collected fractions. If applicable, a UV/VIS spectrophotometer scan will be provided to show incorporation of the correct fluorescent labels into the purified product.
As always we can consult with users on any preferences they have. Contact us by phone or email and we'll do our best to help or answer any questions.
To request HPLC purification simply click on the HPLC button from the list of purification options provided in the Online Order Form.

Modifications

Following is a list of some of the modifications we have placed onto an oligonculeotide:

Also, check out the Glen Research web site www.glenres.com for a list of many other modifications possible. If they sell the precursor compound, we can probably make it for you.

Molecular probes, located at www.probes.com , also carries a wide variety of DNA modifications that we are able to provide for our users.

It is easiest to call or e-mail us if you have ANY questions regarding requests for modified oligos here at the Biotech Center. This extends to questions on pricing as well.

Pricing on oligonucleotide modifications is calculated in a straightforward manner but is variable-the base synthesis cost is exactly what it is normally charged for the scale requested (see table above), and the cost of the modifying reagent from the company is passed directly to the customer. The ultimate cost of the modification per oligonucleotide depends on how many modified oligonucleotides are requested, since once the modifying reagent is prepared for synthesis, it must all be used or discarded. For example, if a user requests a 200 nmol scale synthesis of a single FAM labeled oligo, the cost is $1.25/base plus $165 for the reagent, so that single oligo has a high modifier cost of $165. However, approximately 6 200 nmol syntheses can be accomplished with that same bottle, so if 6 FAM labeled oligonucleotides are requested on a 200nmol scale, the per oligo modifier cost drops to approximately $30. If a user is not in a big hurry, they can request we hang onto the order until additional oligo requests with the same modifier come into the facility. For common modifiers such as FAM we typically receive a slow steady stream of requests so this is one way to save money. Please call if you have any questions about modified oligonucleotide pricing information.

Mass Spectrometry

There is no single quality control procedure that works perfectly on every oligonucleotide, which is why our facility utilizes several approaches. However, one particularly useful quality control tool for most oligonucleotide analyses is mass spectrometry. This procedure is carried out at the Mass Spectrometry facility in the UWBC run by Dr. Amy Harms. The staff here at the Synthesis Facility has been trained in the use of the on-site mass spec machine. We use MALDI-TOF primarily, in which the oligonucleotide is mixed in a matrix, applied to a plate, then subjected to a laser blast which ionizes the product and sends it flying down a tunnel to a detector. The time of flight (TOF) of the ionized product is inversely proportional to the molecular weight of the oligonucleotide. Based on the TOF, the instrument assigns a molecular weight to the various products detected following the laser blasts. Ideally one peak of the correct MW is detected (the expected MW can be easily determined using one of several on-line oligonucleotide calculators-see the URL below for the one we typically use in our facility). Failure sequences, if present, show up as a laddered array of peaks of descending MW, separated by approximately 330 daltons (the MW of a base). In most instances, unless a significant portion of the product resides in failure sequences, the oligonucleotide will work fine for virtually any application.

A user can request to have their oligonucleotide analyzed in this fashion for $5.00 (no free rides here, all the facilities pay to use the other facilities). A hard copy of the data is provided. You can request this on your order sheet by simply clicking yes on the "Mass Spec Option" window. Keep in mind, though, that the oligo will be ready before the data, so you'll have to come back for the hard copy or wait to pick up the oligo. We can also do this on oligonucleotides from your freezer if you want to check the integrity of your stock. If you're having problems on experiments utilizing oligonucleotides, it's one way to possibly rule out that reagent as a problem. Call for more details on what to provide, but typically we would need several ul of a 30-50 ng/ul (5-10 pmol/ul for an 18-mer) solution. An important factor to consider, however, is that very large oligonucleotides (>50 mers), or particularly oligonculeotides with modifications, don't yield great data on the mass spec. Results with such oligonucleotides may be ambiguous and we can make no guarantees about the information gathered. Please call for more information about this.

Oligonucleotide Resources

There are numerous resources on the internet that discuss oligonucleotide chemistry, properties, design, etc.-you can just do a search to find what you need most of the time. However, you may want to check out these web sites to start:

DNA Synthesis Laboratory

Biotechnology Center Contact Information

Custom Oligonucleotide Pricing and Scales

Oligonucleotide Resources