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-15 bis -25 °C lagern","País": "XG","Código": "Lagerbedingun gen (Produkt)","Name": "Lagerbedingungen (Produkt)"},{"Sprache": "de","Wert": "How this product works
light cycler®TaqMan®Master is a ready-to-use reaction mix designed for TaqMan®Recognition format with the LightCycler®Carousel based system.
Contains FastStart polymerase for "Hot Start" PCR which has been shown to significantly improve PCR specificity and sensitivity by minimizing the formation of non-specific amplification products [Chou Q, et al., 1992 and Kellogg DE, et al, 1994] . FastStart Taq DNA Polymerase is a chemically modified form of the heat stable recombinant Taq DNA Polymerase that is inactive at +15 to +25°C and below. The enzyme is only active at high temperatures at which the primers no longer bind non-specifically. The enzyme is fully activated (by removal of blocking groups) in a single preincubation step (95 °C, 10 min) before the start of the cycle. Activation does not require additional operating steps typical of other "warm start" techniques.
the light cycler®TaqMan®Master offers convenience, excellent performance, reproducibility, and minimal risk of contamination. All you need to provide are PCR primers, a detection probe, and your template DNA.
The reaction mix in this kit is optimized for a solid MgCl2Concentration that works with almost all foundation combinations. No need to adjust MgCl2Concentration to amplify different sequences.
The performance of the kit described in this manual is only guaranteed when used with the LightCycler®Carousel based system.
test principle
Hydrolysis probe assays, also called TaqMan®The assays use a single probe containing two labels, a fluorescent reporter dye and a fluorescent quencher. While the probe is intact, the quencher is in close proximity to the reporter dye and quenches the fluorescence of the reporter via fluorescence resonance energy transfer (FRET). When the probe hybridizes to the target sequence, the 5' nuclease activity of the polymerase can cleave the hydrolysis probe, separating the reporter and quencher. As the amount of target sequence increases during PCR, more probe is cleaved and the fluorescent signal of the unquenched reporter dye increases.
Since the principle of hydrolysis probe assays is probe cleavage during PCR, hydrolysis probes cannot be used to perform melting curve analysis. In contrast, HybProbe probes, which consist of two specially designed sequence-specific oligonucleotide probes labeled with different N-dyes, are still intact at the end of amplification and can therefore be used in a melting curve experiment. rear (z.B., for mutation detection or for SNP analysis).
Two-step RT-PCR
the light cycler®TaqMan®Master can also be used to perform a two-step RT-PCR.
In two-step RT-PCR, the reverse transcription of the RNA to cDNA is separated from the other reaction steps and is performed outside of the LightCycler.®Carousel based system. Post amplification and online monitoring are carried out according to the LightCycler standard®Carousel-based system method using cDNA as initial sample material.
One of the following reagents is required for reverse transcription of RNA into cDNA:
- reverse transcriptionist transcriptase
- Transcriptor First Strand cDNA-Synthesekit Transcriptor Alta fidelidad cDNA-Synthesekit Transcriptor Universal cDNA Master
- First Strand cDNA Synthesis Kit for RT-PCR (AMV)
The cDNA synthesis is performed according to the detailed instructions provided with the cDNA synthesis reagent.
Do not use more than 8 μL of undiluted cDNA template per 20 μL of final reaction volume, as larger amounts may inhibit PCR. For initial experiments, Roche recommends running undiluted, 1:10 diluted, and 1:100 diluted cDNA templates in parallel to determine the optimal amount of template.
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| vial/bottle | Tapa | label | function | Catalog number | Content |
|---|---|---|---|---|---|
| 1a | Weiss | Enzymes | After pipetting 10 µL from bottle 1a to bottle 1b:
| 04535286001 | 1 vial |
| 04735536001 | 5 vials | ||||
| 1b | Putrefaction | Reaction mix, 5x conc. | 04535286001 | 3 vials (for 3 × 128 μl) | |
| 04735536001 | 15 vials (for 15 × 128 μl) | ||||
| 2 | colorless | Water, PCR quality |
| 04535286001 | 2 vials, each of 1 ml |
| 04735536001 | 7 vials, each 1 ml |
","Country": "XG","Code": "Content","Name": "Content"},{"Language": "es","Value": "The LightCycler®TaqMan®The Master is designed for research in life sciences. In combination with the LightCycler®A carousel-based system, the kit enables highly sensitive detection and quantification of defined DNA sequences (using suitable PCR primers and detection probes). It can also be used to detect and quantify defined RNA sequences in a two-step RT-PCR (with additional reagents for reverse transcription).","Country": "XG","Code": "Applications","Name " : "Applications"},{"Language": "es","Value": "Ready-to-use Hot Start reaction mix for PCR, with TaqMan®Probes with the LightCycler®Carousel based system.","Country": "XG","Code": "Product description","Name": "Product description"}]}}]}
The performance of the kit described in this manual is only guaranteed when used with the LightCycler®Carousel based system.
Hydrolysis probe assays, also called TaqMan®The assays use a single probe containing two labels, a fluorescent reporter dye and a fluorescent quencher. While the probe is intact, the quencher is in close proximity to the reporter dye and quenches the fluorescence of the reporter via fluorescence resonance energy transfer (FRET). When the probe hybridizes to the target sequence, the 5' nuclease activity of the polymerase can cleave the hydrolysis probe, separating the reporter and quencher. As the amount of target sequence increases during PCR, more probe is cleaved and the fluorescent signal of the unquenched reporter dye increases.
Since the principle of hydrolysis probe assays is probe cleavage during PCR, hydrolysis probes cannot be used to perform melting curve analysis. In contrast, HybProbe probes, which consist of two specially designed sequence-specific oligonucleotide probes labeled with different N-dyes, are still intact at the end of amplification and can therefore be used in a melting curve experiment. rear (z.B., for mutation detection or for SNP analysis).
the light cycler®TaqMan®Master can also be used to perform a two-step RT-PCR.
In two-step RT-PCR, the reverse transcription of the RNA to cDNA is separated from the other reaction steps and is performed outside of the LightCycler.®Carousel based system. Post amplification and online monitoring are carried out according to the LightCycler standard®Carousel-based system method using cDNA as initial sample material.
One of the following reagents is required for reverse transcription of RNA into cDNA:
The cDNA synthesis is performed according to the detailed instructions provided with the cDNA synthesis reagent.
Do not use more than 8 μL of undiluted cDNA template per 20 μL of final reaction volume, as larger amounts may inhibit PCR. For initial experiments, Roche recommends running undiluted, 1:10 diluted, and 1:100 diluted cDNA templates in parallel to determine the optimal amount of template.
", "Country": "XG", "Code": "Start", "Name": "Start" }, { "Language": "from", "Value": "
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "es", "Value": "The LightCycler®TaqMan®The Master is designed for research in life sciences. In combination with the LightCycler®A carousel-based system, the kit enables highly sensitive detection and quantification of defined DNA sequences (using suitable PCR primers and detection probes). It can also be used to detect and quantify defined RNA sequences in a two-step RT-PCR (with additional reagents for reverse transcription).", "Country": "XG", "Code": "Applications", " Name “ : "Applications" }, { "Language": "en", "Value": "Ready-to-use Hot Start reaction mix for PCR, using TaqMan®Probes with the LightCycler®Carousel based system.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" } ] } } ]}