Labs across the world spend a great deal of money on commercially manufactured Taq polymerase for PCR every year. But you don’t have to: Many labs choose to save money by making their own Taq (and Pfu for that matter) for routine applications. This post is not intended to provide a step-by-step protocol for making [...]

Primer design is a very important step while setting up your qPCR assay. If your primers anneal poorly or to more than one sequence, this can significantly impact the quality and reliability of your results. The good news is that primers are cheap, so you can test several different pairs and choose the best pair [...]

In a recent article, I gave some tips about how to obtain good results with sequencing DNA after bisulfite conversion (it contains some tips that apply to the approach described in this article, too). Bisulfite sequencing is a very useful technique if you want to know the methylation status of every CpG in your genomic [...]

Comparing and measuring gene expression is certainly an integral part of research—gene expression patterns continue to show us how different cell networks are regulated, and point to new biological pathways and possible treatments for disease. But one crucial part of gene expression lies in making sure that differences in gene expression are due to gene [...]

For applications such as site-directed mutagenesis, it is often recommended that you use a proofreading polymerase (also known as high-fidelity polymerases) to minimize the risk of introducing unintended point mutations.  But what is a proofreading polymerase?  What makes them different from other polymerases?  And when should you use them?  Read on to learn more… What [...]

Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?

Primer design can sometimes feel like more of an art than a science, and designing the best primer can significantly affect the success or failure of your experiments.  Here are a few tips on optimizing primer design for several different applications: PCR amplification/cloning One of the most common primer-based applications is cloning.  The desired amplicon [...]

Amplify them all and let the melting sort ‘em out. OK perhaps my attitude to analysing sequence variation may be a little flippant but it characterises a lot of my thoughts toward High Resolution Melting Analysis (HRMA). It is now more than a decade since melting analysis to characterise PCR products was introduced. This technique, [...]

Real-time PCR is a technique that is now commonly employed in almost all molecular biology laboratories to quickly answer very specific questions. Northern and Southern blotting are now a thing of the past. No longer do we wait days to know whether a gene is expressed. We can have the answer in 45 minutes! But with the [...]

Whatever the technique, it is essential to have the correct experimental setup to let you verify that you are measuring what you think you are measuring. And PCR is no different – you can’t conclude anything concrete from your results unless you have the correct controls in place. Ian Kavanagh of Thermo Scientific has a [...]