When using the TaqMan® Gene Signature Plates, should I mix the reactions after adding the master mix and template to the wells containing lyophilized primers and probe?

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No, but it is recommended that the plate be centrifuged to draw down the liquid to the bottom of the wells.

Answer Id: 1370

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What are the key features of a TaqMan® MGB probe?

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TaqMan® MGB probes have a nonfluorescent quencher at the 3´ end and a minor groove binder at the 3´ end that increases the melting temperature (Tm) of probe, allowing the use of shorter probes.  For more information on MGB probes, please consult the following manual: "Primer Express v1.5 and TaqMan® MGB Probes for Allelic Discrimination: All PCR Instruments: User Bulletin." You can find a copy on our website by entering this title in the main search box.

Answer Id: 1371

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How can I calculate the concentration of a TaqMan® probe?

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The initial concentration or total yield of your synthesized probe is listed on the data sheet supplied with the probe. TaqMan® probes are sometimes shipped in the lyophilized state, but more often are shipped in solution (1X TE). To create a working stock solution of your probe or primers, you should reconstitute or dilute the probe in the appropriate volume of sterile 1X TE (1 mM Tris, 0.1 mM EDTA, pH 8.0) or sterile, nuclease-free H2O. Detailed instructions on how to calculate the proper volume of solution to add to your probe are provided in our online Tutorial document "Reconstituting and Diluting Primers and TaqMan Probes". You can find a copy on our website by entering this title in the main Search field.

Answer Id: 1512

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Can I replace a TaqMan® probe with fluorescent primers?

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No, fluorescent primers will not function in the same manner as a TaqMan® probe. We do not support any applications using fluorescent primers on our Real-Time PCR platforms.

Answer Id: 1575

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What are the differences in the various types of purification possible when ordering custom oligos?

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OPC: OPC (Oligonucleotide Purification Cartridge) purification is based on the principle of reverse phase chromatography. Oligonucleotides which are to be purified by OPC are synthesized trityl-ON. The trityl (DMT) group at the 5' terminus is hydrophobic, which allows selective retention when the oligonucleotide is added to the cartridge. Truncated failure sequences do not contain a 5' DMT group and are therefore flushed through the cartridge. The retained oligonucleotide is then detritylated and recovered in aqueous organic solvent (20% acetonitrile). This is used to desalt the oligo. It's a way to precipitate the DNA from solution.

HPLC purification: Reverse phase HPLC is used for the purification of oligos. This is the type of purification that will give you the most purity. This type of purification can be requested with large scale oligos.

Answer Id: 1576

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How do I analyze a real time run on an ABI PRISM® 7700 Sequence Detection System in which the run contains reactions that have both TaqMan® MGB probes and TaqMan® TAMRA™ dye labeled probes?

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If the TaqMan® MGB probes are in separate tubes from the TaqMan® TAMRA™ dye-labeled probes, you should analyze the data sets separately. For the TaqMan® TAMRA™ dye-labeled probe, analyze as usual. For the TaqMan® MGB probe you need to uncheck the Quencher box in the Sample Type Setup, and then analyze. This would be done after the run is completed. If you are multiplexing with a TaqMan® MGB and a TaqMan® TAMRA™ dye-labeled probe in the same reaction, it is recommended to leave the quencher as TAMRA™ dye for analysis.

Answer Id: 1772

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