How do I increase my qPCR primer efficiency?
The first thing you could do is to up your TM when performing a 3-Step qPCR. This might help to avoid unspecific primer binding and dimerization. Then you can play with your primer concentrations as well as with the amount of your input cDNA. Or you can design whole new primer sets alltogether.
What is an acceptable qPCR efficiency?
Thus, the PCR amplification efficiency is usually established through calibration or the use of standard curves, and the acceptable amplification efficiency range is 80% to 120% [10].
What is a good primer efficiency?
What is the correct PCR primer efficiency value? Obviously, a perfect primer set will have a primer efficiency of 100%. Therefore, it is recommended that all the primer sets used in your experiment lie between 90 – 110% efficient.
How is qPCR efficiency calculated?
Finally, efficiency is calculated using the equation: E = -1+10(-1/slope). Or use this calculator which does the work for you. Be sure to understand what influences the slope of the amplification curve, as it can otherwise be misleading. Typically, desired amplification efficiencies range from 90% to 110%.
Why is my qPCR efficiency low?
Parameters that affect the efficiency of PCR Your samples may contain PCR inhibitors. Your PCR primer and/or probe design may not be optimal. Inaccurate sample and reagent pipetting. The standard curve may not have been properly analyzed.
How do I increase the sensitivity of my qPCR?
Use Double-Quenched Probes to experience both increased sensitivity and precision in your qPCR experiments. Figure 2. Double-Quenched Probes increase assay sensitivity.
Why is qPCR efficiency important?
The PCR efficiency is one of the most important indicator of the performance of a qPCR assay and is also required parameter for quantitative analysis when fold changes are calculated. Proper usage of PCR efficiency in qPCR analysis requires it is estimated with high precision.
What does high primer efficiency mean?
Amplification efficiency >100% is generally a sign that you are getting primer dimers or that there may be template contamination in one of your reagents.
What does a high primer efficiency mean?
Amplification efficiency >100% is generally a sign that you are getting primer dimers or that there may be template contamination in one of your reagents. Dear Maria, an efficiency higher than 100% could be explained by the presence of inhibitors or an excess of template as Muhammad has pointed.
What affects qPCR efficiency?
Why choose Applied Biosystems real-time PCR platforms?
As a leader in PCR innovation, we offer you the gold standard in real-time PCR instrumentation. With our Applied Biosystems real-time PCR platforms, you get true value with excellent performance, reliability, and world-class support.
Why bother with PCR primer efficiencies?
Why bother with PCR primer efficiencies? Every time you receive a new set of primers, especially when using SYBR Green chemistry during quantitative polymerase chain reaction (qPCR), you should always run a standard curve to calculate the efficiency of your PCR primers.
What is qPCR analysis?
Applied Biosystems® qPCR analysis modules provide an integrated solution for gene expression and genotyping analysis. Data generated from our qPCR platforms can easily be manipulated in the cloud environment. Help optimize efficiency by exclusively using the cloud for data storage once it is collected from the instrument.
How do I calibrate my real-time PCR (qPCR) instrument?
Select your real-time PCR (qPCR) instrument from the drop-down menu, and easily find the plates, optical caps, and optical seals compatible with your instrument. Applied Biosystems Spectral Calibration Kits and TaqMan® RNase P Instrument Verification Plates to help calibrate and verify the performance of your real-time PCR system.