Does Biosearch have a list of all dyes available for DNA labeling?
Biosearch carries a variety of commonly used dyes as well as proprietary dyes.
Please refer to the following link for a complete listing of various dyes/fluorophores we carry at Biosearch:
/download/brochures/bti_bhq_selectionchart.pdf
The dyes indicated in BOLD font are the dyes that we carry at Biosearch. If you have any questions regarding availability of dyes, please contact our Technical Support department at techsupport@biosearchtech.com

How do I determine the amount of diluent to add to my probe to prepare a stock concentration?
We recommend preparing a 100 µM stock solution of your probe. To prepare a 100 µM stock of reconstituted probe: Multiply the “total nmol” value found on Certificate of Analysis by 10. The resulting number will be the volume of diluent (in microliters) to add to your probe. Once resuspended in the appropriate volume of dilution buffer, the probe will be at a 100 µM stock solution.

Tip: Be sure to make aliquots of working concentration of probe to prevent freeze thaw cycles that may degrade the probe. We recommend freezing probes either at -20 ºC or -80 ºC. Probes and primers can be stored in this state for over one year.

How many PCR reactions will I be able to run with my probe or primer? The number of reactions you can run with a given amount of probe or primer is dependent on a few factors, such as input concentration of the primer or probe and the final yield of the synthesis product, which can be variable. If you plan to use your primer for PCR, 1 nmol of product will provide for approximately 100 PCR reactions (when the primer is used at a 200 to 250 nmol concentration in a 50 µL reaction). As for probes, 1 nmol of product is usually enough to run 100-300 reactions but again, this depends on the concentration of probe you are using in your reaction.

I am a beginner at Real Time PCR, do you have any information to help me design my Dual-labeled assay?
There are a few reference articles and resources for information on designing Dual-labeled assays. These articles have general recommendations on oligonucleotide design, cycling parameters and set-up suggestions.

http://dorakmt.tripod.com/genetics/realtime.html

Here’s the link to another helpful resource:
http://www.gene-quantification.info/
This site offers many of great links to articles on Real Time PCR.

Another great resource is the Yahoo! QPCR listserver:
http://groups.yahoo.com/group/qpcrlistserver/

Also, note that we now offer a user-friendly assay software called RealTimeDesign. It’s available on our website, free of charge. For information please inquire at support@biosearchtech.com

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.

http://biosearchtech.com/download/brochures/bti_bhq_handling.pdf

What is the difference between the FAM-BHQ ValuProbe priced at $95 and the $150 FAM-BHQ probe?
The delivery amount for the Valuprobe is 10 nmole, while the standard $150 probe has a minimum delivery of 10 nmole but averages closer to 20 nmole. The other difference is the level of purification. The Valuprobe is purified using Reverse Phase HPLC so that residual fluorescent contamination and other synthesis impurities are removed. The $150 probe is purified by dual HPLC (anion exchange HPLC followed by reverse phase HPLC) which ensures the absolute highest quality in the manufacture of BHQ-quenched dual label probes. The level of purification is dependent on the researchers preference for purification stringency. It’s recommended to use dual HPLC purified probes for multiplex reactions. For more detailed information on the probe quality comparisons, please contact techsupport@biosearchtech.com

When ordering immunochemical conjugates online, I saw that there is a choice for loading. What does this mean? How do I know which loading ratio to choose?
Loading refers to the conjugation ratio, which is the number of hapten groups conjugated to the protein. It is generally accepted that immunization with a lower density hapten-carrier coupled with low doses of antigen preferentially triggers high affinity antibodies. High immunogen doses and high density of hapten on the carrier tend to elicit a higher response since low affinity antibodies are also elicited.

Where can I find usage information and chemical properties for the products you offer?
We typically have product information sheets in the format of pdf documents that contain usage information and chemical properties in the support tab of individual product pages. If for any reason you are unable to find what you're looking for on our website, you can also e-mail us at techsupport@biosearchtech.com

Which dyes are compatible with my thermal cycler?
At Biosearch, we carry both commonly used dyes such as FAM and TAMRA as well as dyes we have developed the Quasar® and CAL Fluor® dyes, because the optics and filters of thermal cyclers vary. We have compiled a chart detailing dye compatibility which can be found here: http://www.biosearchtech.com/multiplexing

You can also download a PDF of our instrument-dye compatibility chart which also includes a dye selection chart.

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).

What are "wobbles"?
When comparing multiple sequences, one may find that alignment reveals no region with sufficient consensus to accommodate a unique single oligo for use as a primer or probe.  In some cases only one or two nucleotides are mismatched.  When designing primers for those regions one may choose to introduce a degenerate site, or "wobble", to compensate for the variability in the target sequence.  Letter codes are used to represent the combination of two different nucleotide phosphoramidites blended at equimolar ratios before coupling at that position in the sequence.  So the end product is a blend of two or more different sequences made simultaneously during one synthesis.
 

Why is it that when a sequence contains a ‘wobble’ it has variable functionality?
Incorporation of ‘wobbles’ into a sequence decreases the effective concentration of each sequence. With increased numbers of ‘wobbles’ the number of distinct species increases exponentially thus decreasing the likelihood that any individual sequence has the desired specificity. Only one species is usually present in a biological sample, therefore a portion of the oligos are not completely complementary to the target and thus, some variability in function is to be expected. To further complicate matters, the individual nucleotide amidites can have different coupling rates. Each time the same ‘wobble’ sequence is synthesized, there is the potential that one species will be produced in preference over another.

Tips:
1) Be conservative. Introduce as few ‘wobbles’ as possible; one trinucleotide ‘wobble’ and one dinucleotide ‘wobble’ or two dinucleotide ‘wobbles’ in two different locations such that there is a maximum of 6 variants in a single oligo; and

2) Increase the concentration of your ‘wobble’ sequence, by as much as two fold, to compensate for the presence of multiple unique sequences.

If you have specific questions regarding minimum yields for a particular probe, please contact Technical Support at techsupport@biosearchtech.com

What formulations of BHQ-1 and BHQ-2 dye do you offer for internal modifications of oligos?
BHQ-1 or BHQ-2 moiety can be attached to the deoxythymidine nucleoside for internal BHQ modifications and should be used when the sugar phosphate linkage must be preserved. When ordering an oligo with a BHQ internal modification attached to a deoxythymidine nucleoside, you can indicate as such using [TBHQ-1] or [TBHQ-2] within your oligo sequence. Here are example oligo sequences showing proper demarcation: ACGT[TBHQ-1]ACGT or ACGT[TBHQ-2]ACGT

Alternatively, BHQ-1 and BHQ-2 dyes can be attached as abasic internal modifications. Take note that these modifications will disrupt the continuity of the sugar-phosphate backbone, and it is not clear how their introduction may impact oligo geometry upon hybridization. You can indicate that you are ordering abasic BHQ internal modifications by inserting “BHQ-1” or “BHQ-2” within your oligo sequence. Here are example oligo sequences showing proper demarcation: ACGT[BHQ-1]ACGT or ACGT[BHQ-2]ACGT

See also: Blog Article - Labeling Oligos with Internal BHQ Dyes

Can Biosearch probes (and dyes) be used for in-situ hybridization (FISH) studies?
Historically FISH probes are cDNA labeled enzymatically with multiple fluorophores. A single synthetic oligo labeled with a fluorophore is not sufficiently bright for visualization under a microscope, and therefore a panel of probes is necessary. Biosearch has manufactured panels of fluorescent-labeled oligos, intended to hybridize in series along the gene of interest. This technique of serial probe binding is called StarFish and is described by Raj et al., in the citation presented below.

Imaging individual mRNA molecules using multiple singly labeled probes. Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Nat Methods. 2008 Oct;5(10):877-9. Epub 2008 Sep 21.