How long is a TaqMan probe?

How long is a TaqMan probe?

18 to 30 bp
TaqMan Design TaqMan probes must be designed (if possible) with a GC-content of 45-65%, a high complexity, no dimer with primers, a high Tm (60-65°C) and a probe length of 18 to 30 bp and probe Tm should be 8-10°C higher than the primers.

How do you design a TaqMan probe?

TaqMan® design parameters used by Beacon Designer™ & AlleleID®

1. Tm Criteria: The primer melting temperature (Tm) should be around 58-60 oC, and TaqMan® probe Tm should be 10 oC higher than the Primer Tm.
2. Length Criteria: Primers should be 15-30 bases in length.
3. GC Content: The G+C content should ideally be 30-80%.

How long should a qPCR probe be?

30 bases
Length: Limit probe length to 30 bases when using dual-labeled probes designed with most common quenchers, as beyond this length quenching ability is decreased.

What is the requirement of TaqMan probe to work?

The TaqMan probe principle relies on the 5´–3´ exonuclease activity of Taq polymerase to cleave a dual-labeled probe during hybridization to the complementary target sequence and fluorophore-based detection.

What is TaqMan MGB probe?

Applied Biosystems TaqMan MGB (minor groove binder) probes are dual-labeled probes used for real-time PCR applications using TaqMan chemistry. TaqMan MGB Probes incorporate a 5′ fluorescent reporter dye and a 3′ nonfluorescent quencher (NFQ).

How do you design a probe?

Design your PCR probes to conform to the following guidelines:

1. Location: Ideally, the probe should be in close proximity to the forward or reverse primer, but should not overlap with a primer-binding site on the same strand.
2. Melting temperature (Tm): Preferably, probes should have a Tm 6–8°C higher than the primers.

How much cDNA do you need for TaqMan assay?

For optimal performance of the assays, use 1 to 100 ng cDNA per 20 µL reaction.

What is TaqMan qPCR?

TaqMan PCR is a type of real-time PCR and uses a nucleic acid probe complementary to an internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second.

What is the quencher in TaqMan?

Schematic of TaqMan (5′ nuclease) assay. The “reporter” (R) dye is attached at the 5′-end of the probe sequence while the “quencher” (Q) dye is synthesized on the 3′-end. A popular combination of dyes is FAM or VIC for the reporter dye and TAMRA for the quencher dye.

What is primer melting temperature?

The recommended melting temperature of PCR primers is usually in the range of 55°C to 70°C and within 5°C of each other. Because of the differences in sequence, length, and composition of the primers, it is often difficult to have similar melting temperatures (Tms) between the two.

Where do TaqMan probes bind?

The probes bind to the polymorphic site of interest during the annealing phase of the PCR reaction. In the extension phase of PCR, the 5′ nuclease activity of Taq polymerase will cleave the 5′ reporter dye from the perfectly hybridized probe, resulting in increased fluorescence.

How much cDNA does TaqMan qPCR have?

How much GDNA do you need for qPCR?

What is the minimum amount of genomic DNA required for analysis using EpiTect Methyl qPCR Arrays? For digestion, we recommend using 0.5-4ug (0.125-1ug per digestion) of genomic DNA for EpiTect Methyl qPCR Arrays and 0.25-0.5ug (0.0625-0.125ug per digestion) for single qPCR assays.

How do I check the TM of a TaqMan probe?

In general, the target Tm of a TaqMan probe is 70 degrees C. Using the Primer Express Software, you can check the Tm of a MGB or TAMRA probe probe. Within the software, go to Primer Probe Test Tool and then copy/paste in your sequence of interest to see the Tm.

What are the differences between TaqMan MGB probes?

TaqMan MGB Probes incorporate a 5′ fluorescent reporter dye and a 3′ nonfluorescent quencher (NFQ). The NFQ offers the advantage of lower background signal, which results in better precision in quantitation. Custom TaqMan MGB probes are HPLC-purified and available with FAM, VIC, TET, and NED reporter dyes. Shorter, more specific probes

Can I design TaqMan® probes for multiplexing?

You can also design TaqMan® for multiplexing up to four sequences, avoiding cross homologies with all probes and primers preventing competition in multiplex reactions. Beacon Designer™ and AlleleID® help you evaluate pre-designed TaqMan® probes or design TaqMan® probes for primers.

What is a fluorogenic probe in a TaqMan® experiment?

While carrying out a TaqMan® experiment, a fluorogenic probe, complementary to the target sequence is added to the PCR reaction mixture. This probe is an oligonucleotide with a reporter dye attached to the 5′ end and a quencher dye attached to the 3′ end.