Which master mix do you choose? How do you know it’s the right one? Choosing the right master mix is an important step when performing your experiment because you want to make sure it is completely optimized for success.
So what is a master mix anyway? It’s a mixture of polymerase, salts, magnesium, dNTPs, and optimized reaction buffer. Your template and primers are the only additions necessary to perform qPCR. Different formulations are good for different situations or applications such as Genotyping, gene expression, MicroRNA quantification, and virus detection
We offer two types of detection technologies when performing Real-time PCR (or qPCR). The first is Applied Biosystems SYBR™ Green, a non-specific master mix DNA binding dye. Specifically, it is based on two hybridization events, the forward and reverse primer The second technology is TaqMan master mix which bases its detection on 5’ nuclease activity. Specifically, it requires three hybridization events: two PCR primers and one probe, with hydrolysis of the probe during extension of the primers.
The first thing to consider when choosing a master mix is what type of application you are working with. For example, let’s focus on Gene Expression. While both technologies can be used, sometimes the experiment is better suited for SYBR® chemistry and sometimes it’s better suited to TaqMan™ chemistry. TaqMan™ chemistry offers the convenience of millions of pre-designed assays and greater levels of specificity but SYBR® offers flexibility for species that aren’t pre-defined which is a value option. SYBR® Green Master Mix includes SYBR® green assays in them, whereas TaqMan require the addition of a label probe. Let’s take a look at what else you have to consider when choosing your master mix. For your gene expression experiment you decide if you need high specificity and a predesigned assay, if so, it’s time for Taqman chemistry, but which one? We can help, but we have a few questions for you: What format are you going to use? 1 step or 2 step ? What mode are you going to run? Fast or standard? Once you select the options that make up your particular experiment, the recommended Master Mix will appear in the online selection tool.
Have more than one experiment? This same selection tool can help you determine which Master mix is recommended for each situation, Whether you have different types of analysis or just different samples types and sizes, we’ll help find the right master mix for you.
If you have more questions about master mix or other qPCR questions, remember, just ask Taqman. Submit your question at https://www.thermofisher.com/ask and subscribe to our channel to see more videos like this.
I’ll see you next time.