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PCR was actually one of the first lab techniques I learned as an undergrad. Despite being sometimes labeled as a pretty basic lab skill, PCR doesn’t always work as expected. This “fickle” success is due to small details or hidden hazards within the PCR workflow that can cause your seemingly uncomplicated experiment to fail. This…
New ways to perform PCR emerge all the time. This speaks for the speed of technological advances, and reflects the ongoing need to keep up with fast-moving research. We all know that PCR’s main purpose is to amplify a stretch of nucleic acids based on sequence-specific primers. Nowadays, a wide range of PCR techniques exist,…
Quantitative PCR (qPCR) uses fluorescent dyes or probes to visualize the amplification of specific DNA sequences as it happens (i.e. in real time). The dyes or probes fluoresce when they bind to newly amplified DNA, and the amount of fluorescence emitted is proportional to the amount of DNA (or mRNA) present in the original sample. By detecting newly synthesized DNA…
Allele-specific expression can occur for various biological reasons, such as gene imprinting, or differential transcription caused by mutations, or single nucleotide polymorphisms (SNPs), or epigenetic alterations. Traditional end-point RT-PCR or qRT-PCR-based methods only detect overall levels of mRNA expression from a given gene rather than mRNA transcripts originating from individuals. If your project requires more…
As is sadly the case in many experiments, site-directed mutagenesis (SDM) does not always work the way we would like it to the first time around. Here are a few tips to help you on your way when trying to troubleshoot a bothersome SDM reaction!
Kary Mullis invented polymerase chain reaction (PCR) in 1985 creating a revolution in molecular biology techniques. But it hasn’t stopped there. PCR has greatly evolved over the years. Today, we stand at a point, where we can clone micro RNAs (miRNAs) in real time! Due to miRNA size (about 18-21 nucleotides long) and varied expression levels,…
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