TaqMan - Molecular Beacons - Fluorescent Molecular Probes
Fluorescent Probes/Molecular Beacons Frequently Asked Questions
(i) In a small-scale synthesis (eg. ~5-10 nmole; 2000-5000 assays), what is the amount of fluorophore-conjugated oligonucleotide typically obtained after purification?
The smallest scale of synthesis offered by Gene Link for Molecular Beacons is at the 200 nmol scale. Synthesis at the lower scale does NOT appreciably reduce cost, as cost of synthesis is NOT directly proportional to the scale of synthesis. Whereas, the higher scale affords Gene Link to comfortably purify the product by polyacrylamide gel and thus provide our customers with a product of the highest quality. Gene Link provides between 7-15 nmols of purified product at the 200 nmols scale synthesis that is sufficient for 2000-5000 assays.
(ii) How are these purified (eg. PAGE or HPLC).
Answered partly above. Gene Link performs and strongly recommends gel purification and believes it to be the Gold standard of purification. All other forms of purification depends on either hydrophobic (Reverse Phase HPLC) or ionic affinity ( Ion-exchange HPLC); both of these techniques are sequence dependent and does not yield the purity achieved by Gel Purification. Gene Link provides a before and after gel picture, i.e of crude and gel purified oligo.
(iii) Is there mass spectroscopic confirmation of the purified product?
Not applicable. Gene Link performs gel purification and supplies a real gel picture. Molecular Beacons provided by Gene Link should have better than 50:1 signal to background ratio. Usually in the range of 200. The purity is greater than 98%.
(iv) Do beacons undergo reverse complement (ie. target) verification and is a close to open ratio analysis performed?
Gene Link currently does not perform these analyses as it considers "after the fact". These analyses do NOT constitute quality of oligo synthesis but rather of design of oligonucleotide. Customers designing their probes should keep the parameters in mind. Gene Link can guide customers to the design of the stem and the loop and the Tm.
(v) What fluorophores are available for conjugation?
The most popular ones are Fluorescein, HEX and TET, Cy5 and Cy3 in that order; others available as post synthesis conjugation are Coumarin, Texas Red, Tamra etc.
(vi) How quickly and in what form are beacons shipped?
Gel purified Beacons are routinely shipped in 4-5 days. These are shipped lyophilized ready to use.
(vii) What type of guarantees could be provided in the event of an inferior or incorrect beacon synthesis (eg. free replacement)?
All Gene Link products are replaced at no charge for not meeting specifications. This means free replacement for the same sequence. This rarely happens, we usually suggest designing a new probe/sequence or to avoid stretches of G" and C's etc.
(viii) Would a free beacon sample be available for testing by our laboratory?
Yes. New customers are offered, "Buy One, Get One Free" of the same fluorophore.
(ix) Would there be on-going technical support in the form of updating us on new developments in beacon synthesis?
Yes. We get to know our customers and talk frequently. The latest development is the wavelength shift, incorporating a different fluorophore in the stem in addition to the 5' end. This is similar to FRET.
Gene Link is not an 'oligo factory" we believe as scientist and researchers that each oligo/probe/gene construction/genotyping etc. etc. is an experiment to be well performed. We believe in quality and consistency and overall in gaining Confidence ourselves in our products and ultimately our customers total confidence in Gene Link products. We hope we have answered most of the frequently asked questions. Please feel free to call (1-800-436-3546) or email firstname.lastname@example.org if you require more information.
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