TCGA Oligo Nucleotide Synthesis

Oligonucleotide Synthesis

Services

TCGA provides quality assured oligonucleotides using state of the art technologies and fast turn around time at affordable cost for your research needs. Stringent quality control of the oligonucleotides done using MALDI-TOF guarantees their good quality. As always every custom DNA oligonucleotide is deprotected and desalted - quality is always guaranteed. Our oligonucleotide synthesis is high throughput and the use of robotics enables us to synthesize a large number of high purity custom oligonucleotides in both tube/plate formats at different synthetic scales.

Custom Oligonucleotides Synthesis	Oligo QC	Ready to use Oligonucleotide
Custom designed Oligos Degenerate Oligos Fluorescent labelled Oligos - 6-FAM - HEX - TET	MALDI-ToF (Sequenom MassARRAY^TM Oligo Check)	M 13 Forward (-40) M 13 Reverse T7 Promoter T7 Terminator SP6 Promoter SK Primer KS Primer
	PAGE



Technology

MerMade-192

	The MerMade-192 is known for its reliability, high throughput, flexibility and ease of use.It has been used to synthesize millions of standard and custom oligos and is flexible enough to fit a wide variety of applications such as gene building, PCR, RNAi, dual labeled probes, micro array, sequencing, antisense, and others. 192 20mer PCR primers can be synthesized in less than 6 hours. Up to 192 oligos (two 96 well plates) in a single run Ability to synthesize in a column or plate format Flexible and easy to use software Run Logging 10 Amidite Ports (more available) Dedicated Thio reagent Bottle Ultra-low dead volume bottle mount for expensive modifiers (>50microliters). Multiple reagent cap adapters as well as kegtype 12 deblock lines for faster operation Sequential or parallel mode for faster plate operation Argon pressure sensor

ABI 3900 DNA Synthesizer

	The ABI 3900 synthesizer is an economical,versatile, high-throughput platform that synthesizes DNA oligonucleotides with fast cycle times and low operating cost. This system is ideal for synthesizing primers and probes, and fluorescently labeled oligos. It synthesizes sequences with lengths greater than 50-mer at scales of 40 nmol, 0.2 µmol, and 1 µmol. It fits compactly on bench top and is ideal for production synthesis.

FAQs

1. How do I order DNA oligos for synthesis?
The oligonucleotide order form is available on our website http://www.tcgaresearch.org or call and we will e-mail or fax it to you. Fill up the submission form and email to oligo@tcgaresearch.org.

2. When can I expect to get the oligos after I order?
Typically, for desalted oligos you can expect delivery within 5 working days. For PAGE purified or fluorescent labeled oligos, delivery time is longer.

3. Do you provide backbone, base or sugar modified DNA oligos?
Currently, we are providing only the commonly used phosphodiester backbone DNA oligos.

4. What about RNA oligos?
No, we do not provide RNA oligos.

5. Can I order oligos of any length?
We can provide oligos as short as 5-bases and as long as 100-bases.

6. Can I order a wobble/degenerate base?
Yes, you can. A mixture of bases is commonly known as wobble or degenerate base. Please find the universal nomenclature in the oligonucleotide order form. When making a wobble, equal amounts of different phosphoramidites are used in one coupling reaction during synthesis. Not all nucleosides are equally incorporated during synthesis, so you might see a slight bias at the wobble position in the final product.

7. Are the oligonucleotides quality checked at TCGA?
Yes, we absolutely do this on a routine basis. TCGA uses MALDI-TOF mass spectrometer for assessing oligonucleotide purity. PAGE is also used to perform quality check for longer oligonucleotides.

8. What kind of purified oligos are provided by TCGA?
We provide either desalted or PAGE purified oligonucleotides.

9. Why is it necessary to purify oligonucleotides?
Due to the large amounts of solvents and the organic reagents used during oligo synthesis, salts in the crude product are unavoidable. The other side reaction during oligo synthesis is the formation of non-full length (n-x) products. The longer the sequence, the more n-x products occur. Therefore purification of oligonucleotides is necessary to avoid potential problems in many applications.

10. I ordered a 50nmole scale oligo. Does this mean that I will receive 50nmole of product?
The scale of synthesis represents the nmole of starting material for the first base of synthesis. The overall nmole yield of full-length oligonucleotide depends on the average coupling efficiency of each base and on the number of couplings (length of oligo). Yields of full-length oligo can vary considerably based on length and type of purification requested. TCGA 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.).

11. 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.

12. Why is the yield sometimes not reproducible?
Obviously, many factors influence the yield. The main reason why you might see different yields for the same oligo is due to the column loading with CPG (controlled pore glass) material used to initiate the synthesis. The minimum loading corresponds to the synthesis scale but fluctuation of the CPG amount and CPG loading can occur which will lead to varying yields.

13. I received my oligonucleotide but there seems to be nothing in the vial. Where is the oligonucleotide?
All oligos are provided in dried form 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 before opening.

14. How should the oligos be resuspended?
For long-term storage we recommend that the oligos be dissolved in a buffer, such as TE (10 mM Tri-HCl, 1 mM EDTA, pH 8.0) instead of just sterilized water. Once resuspended, oligos should be kept frozen at -20°C for longer use.

15. Should I vortex my oligos to resuspend?
We recommend briefly centrifuging your oligo tubes before opening them, this will make sure that the oligo pellet is at the bottom and will not be lost on opening the cap. After adding the TE buffer, vortex to resuspend the oligo.

16. How do I make a 100 µMolar stock solution of my oligo?
TCGA provides quantitative data on your oligo using nmoles and optical density units (O.D.). 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.

17. What does OD mean?
One OD260 (optical density) unit of DNA is the amount of DNA that gives an absorbance reading of 1.0 at a wavelength of 260 nm for a sample dissolved in 1.0 ml total volume of ddH2O, read in a 1 cm quartz cuvette. The OD value is used to determine the concentration of an unknown oligo solution by measuring the absorption at 260nm. 1 OD260 corresponds to approximately 33 µg/ml of single stranded DNA.

18. How stable are DNA oligonucleotides and under which conditions should I store them?
We usually supply DNA oligonucleotides in dried form, since this is less sensitive to degradation by nucleases and more stable for transportation. We recommend you to store this dried oligo at -20 °C or below. Once you have dissolved your oligonucleotides in sterile water or buffer solution, the best way to keep them is to aliquot them into several tubes and store at -20 °C. The sample you are using can be kept in a refrigerator at 4° C for a short time to avoid repeated freezing and thawing of the solution.

19. How should fluorescent oligonucleotides be stored?
The best way to store them is to aliquot the oligo into several tubes, and store the aliquots at -20°C. The sample you are using can be kept in a refrigerator at 4°C for a short time to avoid continuous freezing and thawing of the solution. Additionally, we strongly recommend keeping dye labeled oligonucleotides protected from light.

20. If oligos were left at room temperature for more than a week, would they still work?
Once dried, oligos are supposed to be stable for a long time. Even in solution, they are reasonably stable. Therefore, for the most of cases, without the contamination of materials which can cause degradation of oligos, they should still work well even if they were left at room temperature for more than a week.

Related Links

www.bioautomation.com (for MerMade-192)

www.appliedbiosystems.com(for ABI 3900 DNA Synthesizer)

http://frodo.wi.mit.edu/cgi-bin/primer3 (a tool for primer designing)