We’ve all dealt with them: those expletive-inducing PCR targets that just will not cooperate with regular sample prep workflows. This time of year, we stop thinking of them as “challenging” and see them for what they really are: downright spooky!
Fortunately, Quantabio has your back with even the spookiest of targets. For a great example, consider how we streamline PCR amplification for GC-rich templates. Read the full application note or continue below for highlights.
Poltergeist in the polymerase
Here’s a conundrum: you can’t amplify GC-rich DNA templates without optimization, but the compounds used in that process tend to inhibit polymerase activity. The Quantabio team validated an approach using DMSO or betaine with our repliQa HiFi ToughMix® product for a fast, inexpensive, and efficient method for the PCR amplification of GC-rich templates.
Trick-or-Taq
On its own, repliQa HiFi ToughMix alone overcomes some of the common problems associated with amplifying GC-rich targets. It can typically amplify levels of GC content ranging from 32% to 70% of the overall template. In our test, that was sufficient for amplifying two of the five spooky gene targets we tested, both of which had about 78% GC content. Neither target could be amplified successfully using wild type Taq DNA polymerase.
Not-so-scary enhancers
To tackle the three remaining spooky targets, we evaluated the use of betaine and DMSO in improving the yield and specificity of high-GC templates without inhibiting the polymerase activity of repliQa HiFi ToughMix. We found that when used individually, either enhancer greatly improved amplification for our targets, which had GC content as high as 82.5%.
There you have it — a robust technique for dealing with spooky targets, no ghostbusting required.
Happy Halloween from all of us at Quantabio!
1min
Oct 26, 2023
