ProbeMasterMix (2X) ohne ROX für die qPCR

Question 1

Why do I need a Hot Start Polymerase for a qPCR?

Answer

The Hot Start-modification of the polymerase inhibits its enzymatic activity at low temperatures and prevents non-specific amplifications in the first PCR cycles, which can drastically affect the qPCR results.

Question 2

What is the Ct or Cq value?

Answer

The Ct (Threshold Cycle) value in qPCR is a measure of gene expression and is determined based on the fluorescence curve. The 2nd derivative method is used to determine the maximum change in the second derivative of the fluorescence curve - i.e. the point at which the fluorescence change no longer increases - and thus the exponential amplification is terminated. The cycle in which this is the case is called the "quantification cycle (Cq)". Please note that the calculation of the Cq value can also be done by other methods. It is important that you always use the same method for your experiment.

Question 3

What is ROX?

Answer

ROX(TM) (5-Carboxy-Rhodamin-X) is a fluorescent dye that some qPCR machines use as a passive reference dye. This is done to account for fluorescence variations between wells, which can result from factors like material inconsistencies or imprecise pipetting. In general, it's advisable to take advantage of this normalization option whenever possible. It's essential to determine whether your system requires a low (low ROX) or high (high ROX) concentration of ROX. However, if your cycler lacks a ROX option, there's no need to be concerned. A well-optimized qPCR typically doesn't require additional normalization through a passive dye. Genaxxon provides qPCR master mixes with ROX concentrations that range from none to low or high, catering to all your needs.

Question 4

How can I calculate primer efficiency?

Answer

It's essential to determine primer efficiency for every gene you're analyzing. To do this, create a cDNA dilution series covering a range of 5-6 orders of magnitude and record Cq values for different primer concentrations. You can calculate the primer efficiency from the slope of the resulting line, aiming for it to be within the range of 85% to 110%.

Question 5

Do qPCR plates and foils influence the assay performance?

Answer

Yes, the used plates and foils could influence the assay performance according to several scientific studies (e.g. Reiter and Pfaffl 2008: Effects of Plate Position, Plate Type and Sealing Systems on Real-Time PCR Results, Biotechnology & Biotechnological Equipment, 22:3, 824-828, DOI: 10.1080/13102818.2008.10817561). In general, white qPCR plates (e.g. Genaxxon’s I2017) have a lower light scattering and thus a higher signal strength than transparent qPCR plates (e.g. Genaxxon’s I2004, I2010, I2031). If you have a weakly expressed target or a only small amounts of sample material, we recommend using a white qPCR plate. Beside this, it is essential to use optically clear foils for your qPCR. We do not recommend using “normal” PCR foils as adhesive residues and transparency differences in the material may lead to inhomogeneity during your qPCR assay. Genaxxon offers different qPCR foils: a strongly adhesive foil (I2012) that securely seal the plate immediately, and a pressure-sensitive foil (I2248) that adheres to the plate by applying strong pressure.

Question 6

What are TaqMan Probes?

Answer

TaqMan® probes are hydrolysis probes used in qPCR for measuring DNA levels. Hydrolysis probes are single-stranded DNA oligonucleotides labeled with a fluorescent marker at one end and a quencher at the other. The quencher is a molecule that suppresses the fluorescence of the marker when they are in close proximity. When the probe binds to the DNA target and the polymerase initiates amplification, its exonuclease activity cleaves both the fluorescent marker and the quencher. This action removes the quencher's inhibitory effect. Since this process repeats in every cycle, the strength of the fluorescence signal, measured after excitation, is directly proportional to the amount of amplified DNA.

Question 7

When should I use TaqMan probes, and when should I opt for SYBRGreen?

Answer

Hydrolysis probes (TaqMan) excel in applications such as SNP genotyping, splice variant analysis, and mutation detection using qPCR. In contrast, SYBRGreen-based assays are ideal for gene expression or miRNA expression analysis and offer additional advantages, including quality metrics like melting curve analysis. Several scientific studies suggest that a properly optimized SYBRGreen assay is as effective as a hydrolysis probe (TaqMan) for gene expression analysis (e.g., Tajadini et. al. 2014: Comparison of SYBR Green and TaqMan® methods in quantitative real-time polymerase chain reaction analysis of four adenosine receptor subtypes, Advanced Biomedical Research, 3, 85, DOI: 10.4103/2277-9175.127998). Key factors for success in both cases include selecting appropriate primers, optimizing the qPCR master mix, and ensuring high-quality, non-fragmented cDNA, which is crucial for TaqMan® probes as well. Please not that the performance of a qPCR assay utilizing (TaqMan) probes is heavily influenced by the amplicon length, affecting its robustness, sensitivity, and specificity. In contrast, an assay with SYBRGreen demonstrates greater tolerance for variations in amplicon lengths.

Question 8

How can I check the specificity of my qPCR assay?

Answer

To confirm specificity, you can perform post-qPCR analysis by either running the reaction mix on an agarose gel or, if you're using SYBRGreen, generating a melting curve. These methods will quickly indicate the presence of any unintended byproducts.

Question 9

What controls are essential in qPCR?

Answer

Just like any experiment, it's crucial to include the right controls in every qPCR assay. Depending on the assay format, we recommend the following controls: 1. No Template Control (NTC): For every gene being analyzed, it's important to incorporate an NTC (simply using water instead of a template). NTCs play a critical role in detecting cross-contamination and should be a standard part of every qPCR. 2. Reference Genes (formerly known as Housekeeping Genes): To ensure a robust gene expression analysis, it's a good practice to include multiple reference genes for each sample. These reference genes should exhibit stable expression regardless of any treatment, serving as reference points for subsequent relative quantification. 3. Alternative Positive Control: In cases where you're performing absolute quantifications and not using reference genes, including an alternative positive control is recommended. This control should consistently yield a known result in all scenarios. 4. noRT Control: If you're using reverse transcription (RT) from mRNA as the template, it's advisable to have a noRT control with a sample that lacks the enzyme.

Question 10

Do I need to do a melting curve analysis?

Answer

Please note that a melting curve analysis is only possible when doing a qPCR with intercalating dyes, e.g. SYBRGreen, but not with probes. The melting curve can be used to determine the specificity of a qPCR and to detect any by-products that may occur. It is therefore strongly recommended to perform a melting curve analysis for every qPCR when using an intercalating dye. To generate the melting curve, the qPCR cycler continuously increases the temperature and simultaneously measures the fluorescence. This drops as soon as the melting temperature of the amplicon produced during qPCR is exceeded. Why? When the product melts, the intercalating dye is released, causing the fluorescence signal to drop. Usually, the melting curve is not shown directly as a fluorescence curve, but as a function of the change in fluorescence. A large change in fluorescence is represented as a peak (amplicons denature in this area). Please note that a qPCR always shows only a single peak. Multiple peaks are - with the exception of multiplex assays! - a clear indication of unwanted by-products. These can, under certain circumstances, completely falsify the result and make it unusable. Please note: When changing your mastermix, the peak of a melting curve may shift. Why? The melting temperature also depends on the buffer used in the mastermix, especially on the salt concentration. As different mastermixes may use different buffer, it is quite likely that the resulting melting curve differs.

Question 11

What is the optimal primer concentration?

Answer

The concentration of primers plays a crucial role in qPCR performance and should be carefully optimized. Having a primer concentration that is too high or too low can negatively impact the calculation of the Cq value and may even encourage the formation of primer dimers (see e.g., Mikeska and Dobrovic 2009: Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays, BMC Research Notes, 2, 112, DOI: 10.1186/1756-0500-2-112). As a general guideline, you can initiate with a primer concentration of 100-200 nM. However, it's important to conduct test assays with both higher and lower concentrations to identify the optimal range.

Question 12

How can I calculate primer efficiency?

Answer

It's essential to determine primer efficiency for every gene you're analyzing. To do this, create a cDNA dilution series covering a range of 5-6 orders of magnitude and record Cq values for different primer concentrations. You can calculate the primer efficiency from the slope of the resulting line, aiming for it to be within the range of 85% to 110%.

Question 13

How much template do I need for my qPCR?

Answer

The precise amount of template required for a specific qPCR is contingent on various factors. Excessive template can potentially inhibit the qPCR. In a typical qPCR, the range for template usage per reaction falls between 1 and 10 ng. Therefore, when initiating qPCR optimization, it's highly recommended to establish a template dilution series covering 5 to 6 orders of magnitude. This step is vital for determining the optimal template quantity. This dilution series assists in determining the primer efficiency, the potential for qPCR inhibition by the template, and the verification of whether the template quantity falls within the qPCR's linear range. Once these parameters are established, you can confidently select a template quantity that aligns with the linear range of the qPCR.

Question 14

My qPCR assay is not as specific as it should be. What can I do?

Answer

The specificity of a qPCR assay depends on several factors, including primer concentration, the melting temperature of the primers, annealing temperature and time, and the potential formation of primer dimers. If your qPCR assay lacks the desired specificity, we recommend the following: 1. Reduce the primer concentration. 2. Increase the annealing temperature (while ensuring it doesn't exceed the melting temperature of the primers). 3. Shorten the annealing time. If none of these measures prove effective, consider redesigning the primers, exploring various options for length, melting temperature, and sequence.

Question 15

When using SybrGreen as a fluorescent dye, my quantification is not accurate. What can I do?

Answer

SybrGreen has only a low specificity. It binds to double-stranded DNA during PCR cycles, even to potentially non-specific reaction products. This leads to inaccurate qPCR results with non-optimised protocols. To obtain more specific results, probe-based qPCR should be performed.

Question 16

With the qPCR MasterMix from Genaxxon I get a different Ct value than with my previous MasterMix. What can I do?

Answer

The MasterMixes from different manufacturers have different compositions, including the buffers. Some assays react sensitively to these variations, so that different results are possible. Therefore, a calibration is always necessary when changing the MasterMix. If you have further questions, please contact our technical support: info@genaxxon.com.

Question 17

The qPCR Mastermix does not work with the specified protocol. What can I do?

Answer

The protocols provided by Genaxxon are standard protocols. In order to obtain optimal specificity and amplification, individual optimisation of the experimental conditions is recommended. If you have further questions about this, please feel free to contact our technical support: info@genaxxon.com.

Question 18

Do I need to add ROX™ when using Genaxxon’s qPCR Mastermixes?

Answer

Depending on the qPCR instrument you’re using, you may need to use a passive reference dye, typically ROX, to overcome variations between wells caused by the machine limitations. Therefore, Genaxxon offers qPCR mastermixes with either no or low and high amounts of the passive reference dye ROX™ to ensure compatibility with a variety of qPCR instruments. If you have any problems choosing the right mastermix for your application, please contact our technical support: info@genaxxon.com.

Question 19

Can I use a fast protocol of my instrument settings with Genaxxon’s qPCR mastermixes?

Answer

Genaxxon offers different qPCR mastermixes optimised for fast qPCR assays. Depending on your needs, Genaxxon offers mastermixes for fast qPCR assays with (GreenMasterMix FAST) or without (ProbeMasterMix FAST) a fluorescence dye and with no (No ROX™) or a passive reference dye with different concentrations (Low ROX™ or High ROX™). If you have any problems choosing the right mastermix for your application, please contact our technical support: info@genaxxon.com.

Question 20

Can I easily switch to another qPCR master mix during my project?

Answer

Yes, you can switch to another qPCR master mix during your project or even in the middle of your experiment! You only need to perform a normalization of your qPCR beforehand. Please note that we generally recommend normalization for any qPCR. Also, check if you need to adjust the settings on your PCR cycler when changing the master mix.

Question 21

Can I run multiplex reactions with the Genaxxon qPCR mastermixes?

Answer

Yes, all qPCR mastermixes from Genaxxon can be used for multiplex applications. However, for easy and fast optimization of your multiplex reaction, we recommend using one of our specially designed multiplex mastermixes, e.g., 5X qPCR Multiplex MasterMix (M3024), Multiplex HS MasterMix (2X) (M3013) or the lyophilized LyoPlex Multiplex PCR master mix (M3005).

ProbeMasterMix (2X) ohne ROX für die qPCR

Technische Daten:

Applikation:

Einheitendefinition:

Sicherheits Hinweise / Safety

Klassifizierungen / Classification

Dokumente - Protokolle - Downloads

Zertifikat

Protokolle

Datenblätter

Produktbeschreibung

Allg. Daten 2

Allg. Daten 3

Kategorienliste

Dokumente - Protokolle - Downloads