Hieff Unicon ™ ColorGPS QPCR Master Mix (ingen Rox) _ 11188ES
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Hieff Unicon ™ ColorGPS QPCR Master Mix (ingen Rox) _ 11188ES
Hieff Unicon ™ ColorGPS QPCR Master Mix (ingen Rox) _ 11188ES
Hieff UNICON™ ColorGPS qPCR Master Mix (No Rox) er en præ-opløsning til 2x realtids kvantitativ PCR-amplifikation, med høj fluorescensintensitet, høj sensitivitet og specificitet og højt amplifikationsudbytte. Kernekomponenten, Hieff UNICON™ Taq DNA-polymerase, er varmstartet ved antistofmetode, som effektivt kan hæmme uspecifik amplifikation forårsaget af primer-annealing under prøveforberedelse. Samtidig tilføjer formlen faktorer til at forbedre amplifikationseffektiviteten af ​​PCR-reaktionen og facilitatorer for at udligne amplifikationen af ​​gener med forskellige GC-indhold (30~70%), så den kvantitative PCR kan opnå et godt lineært forhold i en bred vifte af kvantitative regioner. Produktet anvender den blandede farveændringsreaktion...

Color-Traceable qPCR: Zero Mispipetting

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Hieff Unicon ™ ColorGPS QPCR Master Mix (ingen Rox) _ 11188ES

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YeasenSKU: 11188ES03
Størrelse: 1 ml
Pris:
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Hieff UNICON™ ColorGPS qPCR Master Mix (No Rox) er en præ-opløsning til 2x realtids kvantitativ PCR-amplifikation, med høj fluorescensintensitet, høj sensitivitet og specificitet og højt amplifikationsudbytte. Kernekomponenten, Hieff UNICON™ Taq DNA-polymerase, er varmstartet ved antistofmetode, som effektivt kan hæmme uspecifik amplifikation forårsaget af primer-annealing under prøveforberedelse. Samtidig tilføjer formlen faktorer til at forbedre amplifikationseffektiviteten af ​​PCR-reaktionen og facilitatorer for at udligne amplifikationen af ​​gener med forskellige GC-indhold (30~70%), så den kvantitative PCR kan opnå et godt lineært forhold i en bred vifte af kvantitative regioner. Produktet anvender den blandede farveændringsreaktion af forskellige farvestoffer til at overvåge pipetteringsprocessen, hvilket effektivt reducerer forekomsten af ​​pipeteringsfejl.

Funktioner

Pipetteringssporing: Reducer risikoen for mistede/tilføjelsesfejl

Fremragende forstærkning: God linearitet over et bredt lineært område

Høj præcision: Kan præcist skelne skabeloner med en 2-fold koncentrationsforskel

God repeterbarhed: Lille variabilitet mellem brønde i replikatbrønde, hvilket resulterer i bedre repeterbarhed

Stabilt og pålideligt: ​​Reaktionssystemet kan opbevares ved stuetemperatur i 24 timer, og produktet forbliver upåvirket efter 20 fryse-tø-cyklusser

Ansøgninger

Analyse af genekspressionsforskel

Absolut kvantitativ analyse

Specifikationer

Koncentration

2 ×(qPCR-mix)

Detektionsmetode

Farvemetode

PCR metode

qPCR

Polymerase

Taq DNA polymerase

Type prøve

DNA

Applikationsudstyr

Bio-Rad CFX96, CFX384, iCycler iQ, iQ5, MyiQ; MiniOpticon, Opticon, Opticon 2, Chromo4; Cepheid SmartCycler; Eppendorf Mastercycler ep realplex, realplex 2 s; Illumina Eco qPCR; Qiagen/Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000;Roche Applied Science LightCycler 480; Thermo Scientific PikoReal Cycler og andre fluorescens kvantitative PCR-instrumenter, der ikke kræver tilsætning af ROX Reference Dye

Produkttype

Premix til real-time fluorescens kvantitativ PCR

Ansøg til (ansøgning)

Genekspression

Komponenter

Komponenter nr.

Navn

11188ES03

11188ES08

11188ES60

11188-A

Hieff UNICON™ ColorGPS qPCR Master Mix (Ingen Rox)

1 ml

5×1 ml

100×1 ml

11188-B

10×fortyndingsbuffer

1 ml

1 ml

20×1 ml

Forsendelse og opbevaring

Dette produkt bør opbevares ved -25~-15 ℃ i 1 år.

Figurer

Figur 1. Pålidelige resultater kan opnås inden for det dynamiske område.

Ved at bruge 2 µL plasmidskabeloner med en gradient på 10^1-10^7 blev de relaterede gener amplificeret. Hieff UNICON™ ColorGPS master mix kan effektivt detektere et skabelonmængdeområde på 7 størrelsesordener, hvilket opnår god linearitet inden for et bredt lineært område.

Figur2. Kan nøjagtigt skelne skabelonkoncentrationsforskelle på 2 gange.

Ved at anvende 2 µL plasmid fortyndet i en 2-fold gradient som skabelonen blev de relaterede gener amplificeret. Hieff UNICON™ ColorGPS-mastermix kan nøjagtigt løse skabelonkoncentrationsforskelle.

Figur3. Lille variabilitet mellem brønde observeres, hvilket viser bedre repeterbarhed.

Ved at bruge snesevis af kopier af plasmid som skabelon blev amplifikation udført med forskellige fluorescerende kvantitative reagenser i overensstemmelse med procedurerne specificeret i deres respektive instruktionsmanualer. SD-værdierne indikerer, at sammenlignet med andre revers transkriptionsreagenser har det nye produkt mindre forskelle mellem replikatbrønde og bedre repeterbarhed.

Figur4. Mere fleksibel, velegnet til standard eller hurtige procedurer.

I standardproceduren er denatureringstiden og forlængelsestiden sat til henholdsvis 10 sekunder og 30 sekunder, mens denatureringstiden og forlængelsestiden i den hurtige procedure er sat til henholdsvis 3 sekunder og 10 sekunder for at detektere Ct-værdierne svarende til 10 målgener.Resultaterne viser, at Ct-værdierne opnået med det nye produkt i både standard- og hurtige procedurer har en stærk sammenhæng, og det nye produkt kan opnå gode resultater ved brug af den hurtige procedure.

Figur5. Efter at reaktionssystemet er fremstillet, kan det forblive stabilt ved stuetemperatur i 24 timer.

Ved at bruge forskellige gener fra mus, humane 293-celler og Arabidopsis thaliana som mål, blev det efter klargøring af reaktionssystemet anbragt i mørke ved forskellige temperaturer i 24 timer før påvisning. Sammenlignet med øjeblikkelig behandling overstiger detektionsforskellen for forskellige gener ikke 0,5 Ct, hvilket indikerer, at stabile resultater også kan opnås med langvarige operationer.

Documents:

Manualer

11188_Manuel_Ver.EN20241218.pdf

Storage

This product should be stored at -25~-15℃ for 18 months.

FAQ

Please refer to the "YiSheng Biotechnology YiSheng Biotechnology Dye Method Fluorescence Quantitative PCR Mix Selection Table" for details. Click here to learn more and help you choose the appropriate reagents.
For molecular enzyme reagents, simply invert and mix them together, then perform a gentle centrifugation. It is not recommended to vigorously shake to mix them.
Refer to the manual. Ultimately, it is necessary to ensure that after being mixed with the template, the volume is exactly 1×. For example, for 20 μL of cDNA stock solution, add 180 μL of sterile ultrapure water to dilute it 10 times, resulting in 200 μL of diluted template. Take 90 μL of the diluted template and add 10 μL of 10× Dilution Buffer, mix well, and then use it. You can choose not to do this, but it will not affect the result.
You can choose to use the cDNA stock solution or dilute it. Just make sure that the Ct value of the gene you want falls within the range of 15 to 30. Generally, for every 10-fold dilution of cDNA, the Ct value increases by 3.3.
Store in a dark place and the reaction system must not be left open. These two points are essential. It can be placed in a 4℃ refrigerator or at room temperature for 24 hours. Before using, gently shake and centrifuge the sample before loading it onto the instrument. Before loading, carefully check if the tubes or plates have been contaminated.
When designing primers, it is best to cross introns. The design method can refer to "Enjoyable! A single article to understand qPCR primer design, a must-have skill for experimental experts!" Also, validated primers can be selected. The selection method can refer to "Where to find ready-made qPCR primer sequences?"
The possible reasons are as follows:
1. The instrument has not been calibrated for a long time or it is aging. Usually, the instrument needs to be calibrated once a year. It is necessary to have an instrument engineer perform the calibration or replace the aging components.
2. A 10 μL reaction system was used. The normal system is a 20 μL system. Using half of the normal system would result in an excessively low total concentration of Rox, increasing the probability of BadRox occurrence. It is recommended to conduct the experiment using the 20 μL system.
The specificity of qPCR products can usually be determined through the melting curve. If it is still necessary to determine the products through gel electrophoresis, the qPCR product can undergo electrophoresis. Electrophoresis requires Loading Buffer, and the product cannot be directly loaded for electrophoresis.
1. Confirm whether the signal acquisition function is enabled in the program. If a two-step method is used, the signal acquisition should be set during the annealing extension stage.
2. Is the length of the product too long? If the length of the product is set to 500bp or more, this situation may occur. It is recommended that during primer design, the length of the product be controlled within the range of 80-300bp.
3. Are the primers inappropriate? If the primers are not designed correctly, they need to be redesigned. If the primers are correctly designed but seem to be degraded, PAGE electrophoresis can be used to check the integrity of the primers.
4.Template degradation: Prepare the template anew and repeat the experiment.
The analysis is conducted by observing the melting curve. The melting curve shows that the melting peak shape of NTC does not overlap with that of the corresponding gene, and the TM value of NTC is smaller than that of the corresponding gene. In this case, the product corresponding to NTC is a primer dimer. If the melting curve peak shape of the corresponding gene is a single peak, the collection of the signal of the related gene will not be affected. The melting curve shows that the melting peak shape of NTC overlaps with that of the corresponding gene. In this case, the product corresponding to NTC is likely to be the product of the corresponding gene. The system has been contaminated. We need to check one by one whether the water, primers, or reagents have been contaminated. If none of them have been contaminated, it might be aerosol contamination. Based on the obtained amplification data, the ΔCT value is calculated to determine whether the contamination has a significant impact on the system. If ΔCT ≥ 5, it indicates that the contamination has a minor effect and can be ignored.
It indicates the presence of genomic DNA contamination. It is recommended to use an RNA extraction kit that removes the genome for RNA extraction or during reverse transcription by adding a step to remove the genome. Alternatively, when designing the primers, a design across introns should be adopted.
1. If the CT value is less than 30, but the repeatability is very poor, what should we do? Improve the accuracy of sample addition. Avoid adding small volumes and reduce the error of addition. This can be achieved by preparing a large sample system, for example, mixing primers, water, and qPCR mix into a large system or using the method of mixing primers and water into a large system. The large system needs to be mixed evenly. Improve the accuracy of the pipette's aspiration. Pipettes usually need to be calibrated regularly, once a year.
2. What if CT is ≥ 30 but the repeatability is very poor? At this point, due to the low abundance of the effective template, the randomness of collisions between the template and the primers increases, thereby causing a greater difference between the replicons. It is recommended to try adding 2-3 replicons, and then eliminate the data with large differences for data analysis.
The overlapping peaks in the melting curve are located to the left of the main peak. This might be a situation caused by primer dimerization. One possible solution could be to increase the annealing temperature, reduce the primer concentration, or redesign the primers. The overlapping peaks in the melting curve are located to the right of the main peak. a. It is possible that the non-specific amplification occurred due to poor specificity of the primers. The primers need to be redesigned. b. It is possible that the template DNA was contaminated with genomic DNA. It is recommended to use an RNA extraction kit that removes the genomic DNA for RNA extraction, or to add a step of removing the genomic DNA during reverse transcription to reprepare the cDNA template.

Citations

Documents

Brochure
Comprehensive_And_Professional_qPCR_Solution.pdf

Applications

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Forespørgsel

FAQ

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