Ask TaqMan Video Series

For the uninitiated, when we say "technical replicates," we're referring to wells that contain exactly the same amount of the same sample, amplified with the same Assay.

You might be asking yourself "Do I need to do anything special?" First off, size definitely matters when it comes to amplicons in real-time PCR.

Applied Biosystems real-time instruments officially support two different quenchers, the first of which is an oldie but goodie called TAMRA dye. TAMRA dye has been around for a long time, and it's still widely available.

After introducing MIQE guidelines, a set of standards for producing and publishing qPCR data, Sr. Field Application Specialist Doug Rains explains the baselines in Real-Time PCR.

The threshold is a horizontal line in our amplification plot that can be moved up or down on the Y-axis. Its purpose? As we'll see in a minute, it tells the software where to take data. Of course, not all places on the Y-axis are equal.

The causes of multiple peaks in a melt curve when using SYBR Green dye in Real-Time PCR. SYBR Green I chemistry is a free-floating dye. Here's how it works.

But it's normally difficult to see PCR's phases in action. That said, once we do real-time PCR, we can easily visualize its phases. Fortunately, real-time is where PCR phases really matter.

Ask TaqMan answers the question about the advantages and disadvantages of singleplex.

Doug provides a helpful reference document which explains in detail how researchers can quantitatively validate their choice of a control gene.

This is a short video showing how to improve protein expression yields with GeneArt Gene Synthesis and Gateway expression vectors.

Rains explains how large and diverse collection of TaqMan assays comply with these guidelines.

In this video, learn how to make an informed choice about which of the millions of Thermo Fishers' assays are most appropriate for experimental needs.

In this video, Sr. Field Applications Specialist Doug Rains covers the various options that researchers have for performing final qPCR data calculations.

This video looks at improving sample recovery, as well as discusses specialized kits designed to maximize template isolation. Finally, the video explores a novel and highly quantitative strategy for pre-amplification of limited template.

Sr. Field Applications Specialist Doug Rains explores the specific mechanism by which TaqMan® achieves its unparalleled specificity and sensitivity.

In this video, Sr. Field Applications Specialist Doug Rains covers several possible applications that are possible on real-time PCR instruments.

This video also looks at the possible need to dilute RNA prior to reverse transcription. This takes into consideration a host of scenarios including final reaction volume, expression level of genes under study.

MicroRNAs are small and around 22 nucleotide noncoding RNAs that regulate gene expression. Due to their small size, the measurement of miRNAs presents some special challenges.

At the end of the day you want to be able to write up your results and know the changes you see are real -- and not the result of handling or pipeting error.

Did you know that TaqMan assays can also be used for SNP Genotyping? While the assays use the same robust TaqMan chemistry, they contain two probes, and thus analysis is different than when using a Gene Expression assay.

To check the efficiency of an assay, you need to run a template dilution curve in real-time, and then look at the resulting slope of that curve.

How TaqMan Assays amplify target sequences by real-time PCR.

In another Ask TaqMan video, we discussed assay efficiency and how to calculate it. But how does efficiency really impact our experiments, or as Julie Kase at George Washington University asks, why is it important?

If you are studying gene expression by real-time PCR, then you are likely using relative quantitation or comparative Ct method.

There are two primary approaches to reverse transcription for qPCR known informally as two-step or one-step methods. Learn about both methods and when they are recommended.

Copy number variation (CNV) is a type of structural variation that occurs when a DNA segment of 1 kb to several megabases in length is present in variable copy numbers compared to a reference genome