Troublesome Site-Directed Mutagenesis: Troubleshooting Your Experiment for Stubborn Mutations

Troublesome Site-Directed Mutagenesis: Troubleshooting Your Experiment for Stubborn Mutations

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!

How to Prevent False Results in Colony PCR
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How to Prevent False Results in Colony PCR

How to Prevent False Results in Colony PCR Colony PCR saves time and reduces costs by eliminating the need for plasmid purification. However, confounding results abound — but only if you fail to anticipate them. This article outlines the major perpetrators of false results and how to prevent them. For a more general overview of…

Multiplex Ligation-dependent Probe Amplification (MLPA)

Multiplex Ligation-dependent Probe Amplification (MLPA)

Multiplex ligation-dependent probe amplification (MLPA) is a molecular technique developed by MRC-Holland back in 2002. In a nutshell, MLPA is a sensitive technique that allows quantification of nucleic acid sequences, quickly and efficiently. It is performed in many laboratories worldwide, and can be applied to detect copy number changes (like deletions or duplications) of a…

Immuno-PCR:  A Highly Sensitive Method of Immunodetection

Immuno-PCR: A Highly Sensitive Method of Immunodetection

Researchers have relied on immunodetection techniques such as Western blotting, flow cytometry and Enzyme-Linked Immunosorbent Assay (ELISA) for years, but immuno-PCR is a relatively new method. By merging an ELISA with the Polymerase Chain Reaction (PCR), immuno-PCR provides extremely high levels of assay sensitivity. ELISA An ELISA is an assay in which a molecule is…

Primer Validation Was a No-Go – Now What?

Primer Validation Was a No-Go – Now What?

The primers for your gene of interest have finally arrived in the mail, and you’re ready to figure out whether your favorite gene’s expression level is elevated in those precious tissue samples. Only one small last step before you can proceed. Primer validation. This is a standard procedure where you run PCR or qPCR on…

The qRT-rtPCR Control You Should Be Doing, But Probably Aren’t

The qRT-rtPCR Control You Should Be Doing, But Probably Aren’t

Every man, woman, and dog is doing quantitative real time reverse transcriptase PCR (qRT-rtPCR) these days. It’s a great method to measure your favorite transcript’s expression levels. One of the big plusses (like the Swiss flag!) of quantitative PCR in general is its high sensitivity. In principle, it can detect and quantify one molecule of…

Quantifying Allele-Specific Gene Expression Using PCR-Based Methods

Quantifying Allele-Specific Gene Expression Using PCR-Based Methods

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…

Decisions, Decisions: How to Choose the Best qPCR Probe for Your Experiment

Decisions, Decisions: How to Choose the Best qPCR Probe for Your Experiment

Before we go any further, we have to get some things straightened out: RT-PCR versus qPCR versus RT-qPCR. Sooo confusing, amirite?? They all refer to specific molecular biology assays, but the names are unfortunately used interchangeably, which can be awfully confusing for just about anyone. So without further ado: RT-PCR is short for reverse-transcriptase PCR,…

Intercalating Dyes or Fluorescent Probes For RT-qPCR?

Intercalating Dyes or Fluorescent Probes For RT-qPCR?

The unique feature of real-time quantitative polymerase chain reaction (RT-qPCR) is that it associates the amplification of your target gene with a fluorescent signal in a quantifiable manner. Presently, there are numerous fluorescent tool kits/methods to consider when designing your RT-qPCR experiment. However, the two major categories to choose from are fluorescent intercalating dyes and…

Oligo Purification Methods: How, Why and for What?

Oligo Purification Methods: How, Why and for What?

Who amongst us hasn’t had the need for oligonucleotides in an experiment? It is a cornerstone in many procedures and techniques. Depending on the goal, it can be very hard to design just the right oligo for your experiment.  Oligos must have the right length; the right amount of C-G, T-A; they can’t form secondary…

PCR Pitfalls: The Devil is in the Details

PCR Pitfalls: The Devil is in the Details

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…

Divide and Conquer: How to Setup Your First Droplet Digital PCR Experiment

Divide and Conquer: How to Setup Your First Droplet Digital PCR Experiment

Droplet digital PCR?  It’s easy. Because we’re here to guide you through it. We recently introduced you to the principles of digital PCR technology and how it differs from qPCR. In a nutshell, digital PCR is an end-point PCR technology that divides a single PCR into a large number of partitions, and then perform PCR…

How to Choose the Correct Reverse Transcription Method

How to Choose the Correct Reverse Transcription Method

Quantitative Reverse transcription PCR (RT-qPCR) is frequently used in the lab to detect and quantify RNA expression in a sample. The first step of the assay is to convert the labile RNA to its complementary DNA (cDNA) counterpart through reverse transcription (RT). In fact, RT is the first step in a variety of molecular biology…

Small Particles (Things) Matter!- Introducing Nanoparticle PCR

Small Particles (Things) Matter!- Introducing Nanoparticle PCR

There are many different methods and protocols on making your PCR  run more efficiently. I recently came across an interesting PCR method called “nanoparticle” PCR. This method seems to attract a lot of attention, because it enhances a PCR  by a few orders of magnitude. More interestingly, while the enhancement effect has been reported in a…

Reverse Transcription: The Most Common Pitfalls!

Reverse Transcription: The Most Common Pitfalls!

Good quality starting material is king for reverse transcription! Obtaining reliable results in any experiment requires good preparation. We often take reverse transcription for granted, and we don’t always consider that our qPCR might be performing poorly because of problems in that step. Since it’s quite often the reverse transcription reaction itself that causes fuss…

Faster PCR Optimization

Faster PCR Optimization

So, you’ve designed PCR primers to amplify your sequence of interest, and you’re ready to go.  But unless you have a never-ending supply of template, polymerase, and a thermocycler with a gradient function—not to mention a hefty dose of time and patience—you probably don’t want to spend the next week finding the perfect conditions for…

From Revolution to Evolution: Stem-loop Real-time PCR

From Revolution to Evolution: Stem-loop Real-time PCR

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,…

SPUD’s Your Bud When it Comes to Sensitive qPCR

SPUD’s Your Bud When it Comes to Sensitive qPCR

There’s piloting a brand new technique for the first time. Then, there’s jumping through hoops trying to get an established lab technique to work. The former, in contrast to the latter, is expected to be fraught with hardships. Yet troubleshooting an old lab technique that isn’t working anymore, is frustrating at a whole new level….