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| Custom Oligo Synthesis |
Does Biosearch measure A260/A280 ratios of the oligos?
No, we do not measure A260/A280 ratios at Biosearch. A260/A280 ratios are used for measuring protein to DNA concentration following applications in which protein contamination may be an issue, such as extractions from cells. It is not necessary for this ratio to be applied to synthetic DNA analysis as protein contamination is not a concern.
See reference: Validity of nucleic acid purities monitored by 260 nm/280 nm absorbance ratio. Biotechniques. 18(1): p 62-63, 1995.
How do I know what fluorophore to pick for my Dual-labeled probe?
The choice of fluorescent reporter to label your dual-labeled probe(s) depends upon your instrument optics and also the degree of multiplexing you wish to achieve.
If your assays will be amplified separately and not combined together into a multiplexed arrangement, it would be recommended to label each with FAM. FAM is the most commonly used fluorophore and is nicely detected by all real-time PCR instruments.
The optic capabilities of the instrument principally determines the degree of multiplexing and which fluorophores should be used. To determine your selection, visit our Multiplexing qPCR application's webpage.
/assets/bti_bhq_selectionchart.pdf
I have ordered a 5' thiol C6 modification on my custom oligo, what form does that come in and how do I create a free thiol?
Your oligo will be shipped a 5' disulfide with the form HO-C6-S-S-C6-oligo. To reduce it to a free thiol use 100 mM DTT and 0.2 M phosphate buffer pH 8.5 at room temperature for 30 minutes. The product can then be purified with a G-25 column.
I received my oligonucleotide order, but the vial looks empty. Where is the oligonucelotide?
All of oligonucleotides we ship are lyophilized, or in a dry form, unless otherwise specified. Sometimes it may be difficult to see the oligo in the bottom of the vial since it is an opaque pellet. However, be assured that your oligo is in the vial. If you want to be sure that you don’t lose the pellet upon opening the tube, you can briefly spin down the vial in a bench top microcentrifuge.
What are the recommended storage conditions for dual-labeled probes?
Probes should be subjected to minimum number of freeze thaw cycles. Therefore, it is recommended that you prepare and store microvials each having sufficient material for a day’s set of experiment and freeze at -20° C or -80° C. As for long term storage we recommend freezing probes at either -20° C or -80° C. Always protect and minimize your probes from light exposure. We recommend storing probes in amber microtubes or wrapping tubes with foil.
Please let us know if you’d like more information, we can provide a information pamphlet that is a great resource for the entire lab! Email support@biosearchtech.com to request information pamphlet or download from the link below.
/assets/bti_bhq_handling.pdf
What buffer should I resuspend my oligo in?
We recommend preparing stock and working solutions using TE (10mM Tris, 1mM EDTA pH 7.5-8.0) or sterile, DNase-free water. DEPC treated water is not recommended, because it has been known to chemically modify adenine residues and inhibit downstream applications such as enzymatic reactions.
Please note that fluorescent probes are more sensitive than primers and require more care in handling. Rhodamine based dyes, such as TAMRA and ROX are relatively pH insensitive. But for our Quasar 570, Quasar 670 and other dyes such as Cy™3 and Cy5, are physically unstable in acidic conditions and therefore should be stored and used in buffers above pH 7 to prevent degradation.
Oligonucleotides resuspended in water may not dissolve fully in sterile distilled water. Oligos resuspended in water tend to have a pH around 5, and this is often too low for them to completely dissolve. If you resuspend in water, you may need to add NaOH to raise the pH between 7 and 8. If your experiment cannot tolerate EDTA you may want to use Tris-HCl. Remember to store probes protected from light to minimize photobleaching.
What different oligonucleotide purification options does Biosearch offer?
For unlabeled oligonucleotides (primers) Biosearch recommends Reverse Phase Cartridge (RPC) Purification which typically provides 70-75% purity. Contaminants such as truncated sequences, ammonium salts and impurities are removed from the final product. For oligos purified by RPC, the oligos are synthesized with the DMT group left on the final base which allows for separation by affinity of the DMT group to the resin in the cartridge. The principle behind this is that truncated sequences will not have the final DMT group and will not bind to the cartridge and will be washed away. The purified oligo is then eluted from the cartridge. For oligos 50 bases or less the RPC provides high enriched full length product.
For dual-labeled probes, we recommend either the single HPLC or dual HPLC. Single HPLC oligos are processed with Reverse phase HPLC. Dual HPLC oligos are processed with both Reverse Phase and Anion Exchange HPLC.
Reverse Phase HPLC is well-suited to eliminate any fluorescent contamination remaining from the probe synthesis. When left unremoved, this impurity elevates the baseline fluorescence and obscures the detection of probe cleavage. RP-HPLC typically yields purity of products around 70-90% along with the highest yields. The purification technique is similar to Reverse Phase Cartridge but the resins provide greater sample capacity.
Anion Exchange is well-suited to eliminate quencher-only failure sequences resulting from poor reporter or base coupling during the synthesis. When left unremoved, this impurity competes with the probe to find the amplicon and can slightly delay the cycle threshold value.
Dual-HPLC (Reverse Phase and Anion Exchange HPLC) typically results in products with 85-90+% purity.
View our Default and Recommended Methods of Purification Chart for more details.
What is the proper notation if I want to use a deoxyinosine within my oligo sequence?
Within the sequence use the code (dI) including the parenthesis and deoxyinosine will be put at the proper position.
What types of purification does Biosearch offer and how do they compare?
Biosearch offers the following purification procedures:
Desalting: Every oligo is desalted free of charge to remove residual byproducts from synthesis deprotection and cleavage. However, desalting does not remove truncated sequences
RPC (Reverse Phase Cartridge): Cartridge purification provides 80-90% pure (full length) product. This level of purification is recommended for primers or unlabeled oligos.
RP-HPLC (Reverse Phase HPLC): Provides ~90% pure (full length) product. Our FAM-BHQ ValuProbes are RP-HPLC purified.
Dual HPLC (Reverse Phase and Anion Exchange HPLC): Provides ~97% pure(full length) product and is the method of choice when more stringent purification processes are needed for applications such as multiplexing reactions.
Please Note: The purity percentages given are general guidelines and these values can vary depending on the sequence of the oligo.
Why does the synthesis scale ordered for my oligonucleotide not correspond with the final yield?
The synthesis scale and final yield are not the same because there are several steps in the synthesis of probes and primers. These steps include coupling of each base, cleavage of oligonucleotide from solid support and purification steps. The combination of these steps cause the final yield of the oligonucleotide to be less than the synthesis scale (starting material)
If you have specific questions regarding minimum yields for a particular probe, please contact Technical Support at techsupport@biosearchtech.com
Why is BHQ-3 not an option when selecting Quasar or Pulsar dye labels?
Although we do offer BHQ-3 as a modification for oligos, we do not offer it paired to Pulsar 650, Quasar 670 or Quasar 705. For those dyes, we recommend BHQ-2. We do have data showing that BHQ-2 would be the optimal quencher to use. If you look on the Biosearch website under the Dual-labeled Probes, you will see that we automatically paired Pulsar 650, Quasar 670 or Quasar 705 with BHQ-2:
http://www.biosearchtech.com/store/product.aspx?catid=224,171,40
We generally do not recommend using BHQ-3 for Dual-labeled Probes since we cannot guarantee its results (minimum yield, functional performance, etc). It may be somewhat misleading when you look at the spectra of BHQ-3 compared to Quasar 670 because it is illustrating the traditional model of quenching and spectral overlap. It is possible that the principle quenching mechanism is not FRET but rather static quenching. A hydrophobic attraction between the fluorophore and quencher can promote the formation of an intramolecular dimer, which is non-fluorescent.
Scientists at Biosearch have characterized this quenching mechanism in several papers.
Why were my oligonucleotides shipped at room temperature?
At Biosearch we ship all lyphophilized oligos overnight. Dried down oligos are very stable at room temperature. If you have requested your oligos in solution and they were not shipped frozen, contact our technical support team by email at techsupport@biosearchtech.com or call 1-800-436-6631.
Does Biosearch have a list of all available DNA modifications?
Biosearch offers a comprehensive selection of fluorescent and non-fluorescent modifications for 5’, 3’ and internal labeling.
For a complete listing of modifications available at Biosearch, see link: http://www.biosearchtech.com/products/custom-oligonucleotides/modifications.aspx
If you have any questions about modification availability please contact our Technical Support department at techsupport@biosearchtech.com
Is there a limit to the length of oligos Biosearch will synthesize?
Biosearch will synthesize oligos up to 120 bases long.
Are unlabeled primers modified with a 3' phosphate?
Our unlabeled primers are not synthesized with phosphate modifications unless requested and specified as a custom primer upon ordering. Unlabeled primers are synthesized with terminal hydroxyls (5' and 3'). Phosphate modifications at the 5' and 3' ends are available as custom requests. For pricing of custom modifications contact our customer service department at info@biosearchtech.com
Does my oligonucleotide have a phosphate on the 5’ or 3’ end?
All of our standard 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. Dual-labeled fluorescent probes will not be extended as long as there is a quencher at the 3’ end. As for probes with internal quenchers, a 3’ phosphate will be added to eliminate undesired extension.
Do you make primers that are unextendable?
Yes, in order for a primer to be unextendable, a 3'-modification is commonly used. A phosphate modification synthesized at the 3’ terminus will render the primer unextendable. Dual-labeled fluorescent probes will not be extended as long as there is a quencher at the 3' end. For probes with internal quenchers, a 3' phosphate can be added to eliminate undesired extension. Please indicate that you would like a custom oligo synthesized with a 3’ phosphate modification synthesized.
Does Biosearch synthesize Minor Groove Binder DNA probes?
No, we do not synthesize MGB probes; however, we do sell a new and advanced probe technology for qPCR, the BHQplus probe. This novel, compact probe permits the design of shorter oligos that can detect more difficult targets such as AT-rich regions and SNPs.
Are Locked Nucleic Acids (LNA) available through Biosearch?
No, we do not synthesize oligos with LNA modifications; however, we do sell new and advanced probe technology for qPCR, the BHQplus probe. This novel, compact probe permits the design of shorter oligos that can detect more difficult targets such as AT-rich regions and SNPs.
What is the difference between a BHQ and BHQplus Probe?
BHQplus probes use a duplex stabilizing technology that allows for shorter oligonucleotides with relatively high melting temperatures that can detect difficult targets such as AT-rich regions and single nucleotide polymorphisms as compared to BHQ probes that are mainly used in standard qPCR and FRET applications.
What is the difference between static quenching and FRET?
Static (contact, ground state) quenching involves formation of a reporter-quencher dimer. The intramolecular dimer effectively decreases the concentration of the fluorescent reporter by creating a new, nonfluorescent reporter-quencher dimer with a unique absorption spectrum. FRET is a dynamic quenching mechanism that does not affect the probe’s absorption spectrum. Hybridization of the dual-labeled probe to its target or nuclease activity disrupts the reporter- quencher dimer, allowing the reporter to return to the state allowing fluorescence to occur.
Can I use the Nanodrop to measure the concentration of synthetic oligos?
YES. Nanodrop technology can be used to measure the concentration of individual synthetic oligos using each oligo’s unique analysis constant. By default, the Nanodrop equipment uses a value of "33" as a general constant for all single-stranded DNA, which is inappropriate for synthetic oligos. Oligos purchased through Biosearch Technologies arrive with data sheets containing the extinction coefficient and molecular weight of each oligo. These numbers are used to calculate the analysis constant needed for Nanodrop concentration calculations. Use the formula below to calculate the Analysis Constant(AC):
AC = (1/extinction coefficient) x (Molecular Weight(protonated)) x 1000
= AC in micrograms per OD260nm
When measuring labeled oligos, we have determined through internal research that the Nanodrop's linear range of detection is much more limited than advertised. For oligo stocks in the 100 µM range, the Nanodrop will record an apparent concentration that is significantly below the actual concentration. We recommend diluting such stocks 25-fold to achieve a concentration in the range of 4 µM, and enable accurate measurements.
