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 [...]

Calculations can be the bane of laboratory work.  Therefore, I have always looked for easy methods for getting the math done. Here are several different ways to calculate primer concentration depending on the starting material.  For all calculations, let’s assume we have 22 nmol of a DNA primer containing 16 bases. From lyophilized powder Primers [...]

During my postgraduate studies, I did literally one PCR reaction with a pre-optimised protocol on a not especially difficult template. So my karma came back with vengeance, when as a part of my first postdoc I had to amplify a template containing a 35 bp-long GC-rich stem-loop, which proved to be extremely difficult. This was [...]

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?

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 [...]

One of the very first things you need to do when getting set up for quantitative PCR (qPCR) is to determine the efficiency of the assay because knowing the assay efficiency is critical to accurate data interpretation. And you have to do this every time you design and purchase a new primer pair. Ideally, the [...]

One of the great features of PCR is its excellent sensitivity as we know. And many articles describe real-time qPCR as an added leap forward in that sensitivity – to the point where it has become a standard feature of a new assay description Indeed, I’m currently developing a new qPCR assay to replace a [...]

The problem of amplification of DNA templates containing high GC content continues to annoy researchers despite the many strategies developed to overcome this issue. Betaine is the most common additive used to enhance amplification of GC rich sequences because of its ability to dissolve secondary structure that blocks polymerase action.  But betaine does not solve every [...]