Imagine you are preparing your samples for PCR on a hot summer day. You have already mixed all the necessary components together, now you just have to insert your samples into the PCR machine. Suddenly your co-worker shows up and says that they need only a few minutes of your time to discuss a very important matter. As per usual a few minutes then turns into fifteen minutes. Finally, you turn to your samples and insert them into the PCR machine. A little while later it’s time to analyze the results, but for some reason, they are messy and don’t make sense. You sigh since you have wasted a perfectly good summer day, but gotten nothing out of it. So how to make sure this doesn’t happen again?
First, let’s talk about what went wrong. Presuming that there was no contamination and all the primers and PCR mix components were working properly, there was only the issue of letting your samples sit at room temperature for fifteen minutes. During that time your primers probably randomly bound to DNA templates or to each other forming primer dimers and starting non-specific amplification, because there was nothing to stop them from doing that. In the end, you ended up with lower yield and non-specific results, since part of the PCR efficiency went to amplifying unnecessary segments [1].
To avoid that the easy answer is using the hot start PCR technique. With this technique, you don’t have to worry about leaving your samples on the table while talking to a colleague or worry that once you get to the last samples the first ones might already be messed up. The main idea behind a hot start is that the reaction needs a hot start to work [1][2]. No non-specific amplification will occur during the reaction setup since the specially modified DNA polymerase won’t work until heated at 95 °C in the PCR machine [1][2]. So using the hot start PCR technique means that not only can you work calmly on your samples at a room temperature, but you will also end up with higher yields, better specificity, and sensitivity [1][2].
For your convenience, most Solis BioDyne PCR and qPCR mixes already contain reagents required for the hot start. We use chemical and oligo hot start methods to keep our enzymes inactive during reaction setup. In addition, due to Stability TAG technology, all our enzymes and master mixes have enhanced stability at room temperature with no activity loss for up to 1 month, so you don’t have to worry about not using ice while preparing your samples. Also, be sure to check out our SolisFAST® line products that are not only thermostable but so fast that you can repeat your perfect result and still have time left to enjoy the summer.
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.