PCR Cloning: Answers to Some Frequently Asked Questions

Posted on by Kelly Grooms

eh1Q: What is the easiest way to clone PCR Products?

A: The simplest way to clone PCR Products is to amplify the product using thermostable polymerases such as Taq, Tfl or Tth polymerase. These polymerases add a single deoxyadenosine to the 3´-end of the amplified products (3´-end overhang), and can be cloned directly into a linearized T-vector.

Q: What if my DNA polymerase has 3´ to 5´ exonuclease activity (i.e., proofreading activity) that removes the 3´-end overhang?

A: To clone PCR products that have been amplified with a polymerase that have proof reading activity into a T-vector, you will need to perform an A-tailing step using Taq DNA polymerase and dATP. Blunt ended restriction digest fragments can also be A-tailed using this method. The method below uses GoTaq Flexi DNA Polymerase (comes with a Mg-free reaction buffer), but any Taq DNA polymerase can be used.

Set up the following reaction in a thin-walled PCR tube:

1–4µl purified blunt-ended DNA fragment (from PCR or restriction enzyme digestion)
2µl of 5X GoTaq Reaction Buffer (Colorless or Green)
2µl of 1mM dATP (0.2mM final concentration)
1µl GoTaq Flexi DNA Polymerase (5u/µl)
0.6µl of 25mM MgCl2 (1.5mM final concentration)
Nuclease-free water to a final volume of 10µl

Incubate at 70°C for 15–30 minutes in a water bath or thermal cycler.

Q: What is a T-vector, and why are they used for cloning PCR products?

A: T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.

For a complete PCR Cloning protocol, Visit the Cloning Chapter of the Promega Protocols and Applications Guide.

Friday Fun: A Cartoon Limerick for the Poetic Scientist

Posted on by Promega

Your Promega Connections bloggers thought we would inject a little levity into the day. So we are sharing this Ed Himelblau cartoon limerick.  Are any of our readers poets? Do you have a couplet, haiku or science-related limerick you can share with us?

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Tracking the Beginning of a Pathogenic Bacterial Infection

Posted on by Sara Klink

Yersinia pestis by U.S. Center for Disease Control [Public domain], via Wikimedia Commons.
Understanding the course of a pathogenic infection involves not only understanding what ultimately kills the host or how the bacterium or virus enters the body but also how it establishes itself in the host organism. What is the receptor that allows a virus to enter the cell? Which cells does a bacterium first target or how does it evade an immune response? While other studies of bacteria like Yersina pestis have looked at imaging the bacterial burden in model mice, questions remain about how this bacterium gets from the skin after an infected flea bites to the draining lymph nodes, where the bacteria replicate and enter the bloodstream and infection becomes fatal. A recent PLOS Pathogens article examined how the nonmotile Y. pestis disseminated itself starting from a tiny innoculation mimicking a flea bite on a mouse ear and following pathogen interaction with the host from skin to lymph node. Continue reading “Tracking the Beginning of a Pathogenic Bacterial Infection”

Choosing the Best Luciferase Vector for Your Experiment—Now Made Easier with the Vector Selector

Posted on by Michele Arduengo, PhD

4621CAGenetic reporters are used as indicators to study gene expression and cellular events coupled to gene expression. They are widely used in pharmaceutical and biomedical research and also in molecular biology and biochemistry. Typically, a reporter gene is cloned with a DNA sequence of interest into an expression vector that is then transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. A good reporter gene can be identified easily and measured quantitatively when it is expressed (in the organism or cells of interest).

Bioluminescent reporters are ideal for these types of studies because they have a number of important features including:
• Measurements that are almost instantaneous
• Exceptional sensitivity
• A wide dynamic range
• Typically no endogenous activity in host cells to interfere with quantitation

However, one factor that is critical for the success of a bioluminescent reporter assay is the vector.

At Promega we offer several different luciferases as reporters, and the genes for those luciferases are available in a variety of vectors. The vectors may vary in the promoters used or the presence or absence of sequences for rapid degradation. Often seemingly small changes in the vector can make a big difference in the suitability of the vector for a given experimental system. Do you need a reporter with a short half-life to detect rapid changes in gene expression? Are you studying a specifically localized protein? Do you wish to perform a transient or stable transfection?

To make finding the best reporter vector for your experimental system easy, we have developed the Luciferase Reporter Vector Selector. Using this online tool, you can narrow the choices of available vectors by promoter type, application (in vivo imaging, cancer pathway analysis, etc), availability of selectable marker, and type of luciferase.

So, as you design your luciferase reporter experiment, keep in mind this handy tool to help you choose the best luciferase vector for your needs.

How Confident are You in Your Mass Spectrometry Data?

Posted on by Gary Kobs

Data generated by scientific instruments and decisions based on that data depend on optimal instrument performance. Clinical assays rely on mass spectrometric (MS) data for accurate results so that correct health related results are gained and appropriate results-based decisions are made. However, there are no generally agreed upon tools nor performance standards for mass spectrometry. Furthermore, while several software tools exist that serve to assist  with the analysis of instrument performance, a dedicated reagent software package has yet to be created. For optimal liquid chromatography (LC) performance, parameters like retention time, peak width and peak heightare typically reported. Commonly monitored MS parameters include mass accuracy, mass resolution, signal-to noise, sensitivity, limit of detection (LOD), limit of quantitation (LOQ) and dynamic range.

The 6 × 5 LC-MS/MS Peptide Reference Mix is designed for use in method development and optimization,and for routine liquid chromatography (LC) and mass spectrometry (MS) instrument performance monitoring. The product is a mixture of 30 peptides: 6 sets of 5 isotopologues of the same peptide sequence. The isotopologues (Figure 1) differ only by the number of stable, heavy-labeled amino acids incorporated into the sequence. The labeled amino acids consist of uniform 13C and 15N atoms. Each of the isotopologues is indistinguishable chemically and chromatographically. However, since they differ in mass, they are clearly resolved by mass spectrometry.

Figure 1.
Figure 1.

The isotopologues of each peptide are present in a series of tenfold differences in concentration or molar abundance. If 1pmol of the mixture is loaded onto an LC column, the next lighter isotopologue would be 100fmol, the next 10fmol, the second lightest 1fmol, and the lightest 100amol. This range allows assessment of the instrument’s dynamic range and sensitivity from a single run.

Peptides with a wide range of hydrophobicities were chosen to enable reporting of LC column performance. The most hydrophilic peptide gives users a tool to optimize the capture of hydrophilic peptides that might be difficult to capture otherwise, but that are too precious to use for method development.

To assist in data processing, a complementary software tool, is provided, the 6 × 5 LC-MS/MS Peptide Reference Mix Analysis Software (The PReMiS™ Software). The PReMiS™ Software produces a tabular report of calculated instrument parameters, graphical analysis of linearity curves as well as reporting the history of user-selected parameters such as LC retention time, peak height and mass accuracy. If the laboratory has a collection of instruments, there is also an option to compare parameters across instruments.

A Whole New Meaning to the Phrase “Catching Cold”

Posted on by Terri Sundquist

ThermometerEvery day we are bombarded by potential contagions: whether a physical ailment such as measles or influenza or something as seemingly harmless as a yawn or popular Internet meme. For better or worse, emotions can be contagious too, passed on from one person to another through verbal and nonverbal cues, with or without the awareness that we are being affected by another person’s emotional state. In many cases, the only route for such transmission is observation. For example, who hasn’t felt better after watching an uplifting film or cried while watching a sad movie. In the lab, scientists have determined that levels of the stress hormone cortisol increased in individuals undergoing a stress test but also in passive observers who watch the stress test through a one-way mirror or on a television screen. Often, the magnitude of the observer’s response is affected by how well he knows or can relate to the person.

As reported in a recent PLOS ONE article(1), we now know that even a physiological response to cold temperatures is readily transmissible from one person to another, although many of us who live in northern climates probably knew this long before any scientific study: Watching children playing in the snow or someone shoveling snow can often send a chill through us even though we are watching from a heated building. However, a group of scientists in the UK and Germany was not satisfied with anecdotal evidence of temperature contagion, as they named this phenomenon. They did the experiment and generated the data.

Continue reading “A Whole New Meaning to the Phrase “Catching Cold””

Biotechnology Ice Breakers: A Few Conversation Starters

Posted on by Amy Prevost, PhD

quiz pictureThe biotechnology industry is one of the most dynamic out there – in fact, it never stands still! For non-scientists this can be intimidating. For scientists, it can be challenging to explain what we do in ways that non-scientists can understand and appreciate.

Scientists have made great strides in improving our ability to use molecular processes to our advantage, from discovering the basics of how to isolate and manipulate DNA to gaining an understanding of how genes direct the creation of proteins in cells.  It’s clear that there is a lot we can contribute to the scientific literacy of the general public.

In this spirit, we’ve designed a short quiz for both non-scientists (you may learn something new) and scientists (you may find it useful for engaging in conversations with your non-scientist friends and family members).  Spoiler alert: answers are provided. Continue reading “Biotechnology Ice Breakers: A Few Conversation Starters”

Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast

Posted on by Kelly Grooms

Starfish BlueA killer is lurking in the waters off the pacific coast. Silent and lethal, it leaves its decimated victims in tidal pools. They first began to appear in the early summer of 2013. Limp and curled, missing some or all of their limbs, the bodies were little more than globs of slimy tissue.  They were hardly recognizable as what they once were—Sea Stars. Continue reading “Death in the Stars: A Virus Decimates Sea Star Populations Along the Pacific Coast”

MicroRNAs as Circulating Biomarkers

Posted on by Radhika Ganeshan

12097693_lMicroRNAs (miRNAs) are short strands of RNA averaging between 19-24 nucleotides in length that were first discovered in C.elegans and subsequently shown to exist in species ranging from algae to humans (1). Speculated to be merely “junk” more than a decade ago, miRNAs have emerged as powerful regulators of a wide array of cellular processes because of their influence on gene expression at the posttrancriptional level. Dysregulation of these miRNAs is also associated with life-threatening conditions such as cancer and cardiovascular disease, which points to a potential use of miRNAs in diagnosis and treatment. Recently, it has been demonstrated that miRNAs are present in circulating blood plasma, protected from degradation by inclusion in lipid or lipoprotein complexes. This opens up the possibility to exploit miRNA as a useful diagnostic tool in clinical samples. Continue reading “MicroRNAs as Circulating Biomarkers”

2 Ways to Save Your Single Reporter Data

Posted on by Kyle Hooper

Reporter assays using a single reporter, be it from a stable cell line or transient transfection, can benefit from normalization. Obviously, we are not talking about adding a second control reporter but normalizing to the number of live or dead cells in the well.  Two cell health assays, CellTiter-Fluor™ Cell Viability Assay and CellTox™ Green Cytotoxicity Assay, are ideally suited for multiplexing with reporter assays. 

Continue reading “2 Ways to Save Your Single Reporter Data”