To help you get into the multiplexing workflow efficiently, there are a few key points to consider:
Figure 1. SolisFAST® Probe qPCR Mix (ROX) was used in a 4-plex probe-based qPCR with fourteen fold serial dilutions of human gDNA (40 ng –40 pg per reaction, three replicates at each concentration). qPCR was performed on a QuantStudio™ 6 Flex qPCR cycler (Applied BioSystems™) using ROX dye for normalization.
Using regular qPCR mixes in highly multiplexed assays carries a number of risks, such as late Ct values, low efficiency, low sensitivity, and low fluorescence. As regular qPCR mixes do not have a sufficient amount of components for amplifying more than 2 targets simultaneously, you are running the risk of getting false-negative results. Using specifically designed multiplex mixes will greatly increase your success.
For fast cycling speed and up to 5-plex reactions:
For standard cycling speed and up to 4-plex reactions:
By using the right product designed for multiplexing, you can rest assured you will enjoy similar sensitivity to singleplexing with a greatly reduced workload.
Figure 2. HOT FIREPol® Multiplex qPCR Mix (Purple) was used to perform 4-plex or single-plex probe-based qPCR with five tenfold serial dilutions of human cDNA (cDNA concentration ranges from 200 ng to 20 pg per reaction). Reactions were performed with Applied BioSystems™ QuantStudio™ 6 cycler using Purple dye for normalization.
Lyophilization, also known as freeze-drying, is in simple terms a water-removal process that increases product stability and preserves its functionality. Our new SolisFAST® Lyo-Ready qPCR Kit with UNG represents an optimized lyophilization-compatible qPCR solution to enhance the simplicity, convenience, and speed of diagnostic and applied testing.
The running joke with PCR is that if something can go wrong, it will go wrong. Quite often it’s even impossible to determine why some samples turned out fine while the others did not. In a situation like this, it would be amazing to know some trick or a secret to avoid spending all the time and resources to do the experiment again. Here are a few we are willing to share so that you could find love for PCR.
In research, every day different methods are used to discover something new, whether it is a new disease, medicine, or something else. Often these methods were developed long ago and are confirmed to be doing what they are supposed to do. However, as technology develops so do new methods. This is exactly what Professor Steven Williams’ lab is doing at Smith College – developing new methods to be used in research and diagnostics.
As an alternative to PCR, the loop-mediated isothermal amplification (LAMP) reaction has been developed for DNA detection. The LAMP test is fast, simple, and sensitive.