What’s The Problem With Ampicillin Selection?

What’s The Problem With Ampicillin Selection?

Resistance to the antibiotic ampicillin is commonly used as a selection marker for plasmids in gene cloning and protein expression in E.coli and other bacteria. While it can be incredibly useful tool, there can be problems using this selection marker that you need to be aware of if if you plan on using it. This…

Antibiotics Used in Molecular Biology

Antibiotics Used in Molecular Biology

Antibiotics are used in a wide range of techniques in molecular biology including molecular cloning and are important for treating pesky mycoplasma contamination in cell cultures. They can also be used to maximize your plasmid yields by reducing protein synthesis, in certain circumstances. The aim of this post is to provide an easy reference to…

Top Tips for Troubleshooting In Vitro Transcription 

Top Tips for Troubleshooting In Vitro Transcription 

The Phobia of RNases My first experience of troubleshooting in vitro transcription came when I was synthesizing RNA In-Situ Probes for the first time. A lab mate ominously warned me that I had just returned to lab after a bout of flu and that meant I’m a walking talking factory of RNases. I went ahead…

Faster Ligations: PEGing down the Secret

Faster Ligations: PEGing down the Secret

Overnight ligations are inconvenient — especially when they fail. Luckily, there’s a straightforward way to faster DNA ligations. This article highlights the secret ingredient to faster ligation reactions and offers some tips and caveats on its use. For a general overview of DNA ligations, see here and here. Buy a Quick Ligation Kit The most…

How to Set up Your Elution Experiment

How to Set up Your Elution Experiment

What do DNA mini preps and protein immunoprecipitation experiments have in common? They start differently, but they end with the same, critical stage – elution. But what exactly is elution, and what is the point? The Terminology First, let’s start with some basic terminology: Elution – extracting one material from another by washing with a…

CPEC– a Quick and Inexpensive Cloning Strategy

CPEC– a Quick and Inexpensive Cloning Strategy

Cloning Strategies – a Whole Lot of Options to Choose Molecular cloning has come a long way from simple restriction digestion-ligation cloning strategies to a large number of highly efficient alternatives. Broadly classified, cloning techniques can be divided as sequence dependent and sequence independent strategies. Sequence-dependent strategies are based on restriction digestion-ligation techniques or site-specific…

Show Your Molecular TALEN(T)

Show Your Molecular TALEN(T)

Introduction Did you know that the idea of using genetic engineering to ameliorate certain human diseases was viewed as ‘science fiction’ only 10 short years ago? While cell mutagenesis studies and genetic knockout experiments were feasible before genetic engineering, they were not very reliable. Indeed, due to the random and imprecise nature of these older…

NGS Target Enrichment Strategies
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NGS Target Enrichment Strategies

Next-generation sequencing (NGS) has ushered in a new era of understanding of both the inner workings and the function of the genome. NGS allows researchers to look at traits—including diseases—that are linked to differences or mutations in an individual’s genes. Since only about 1% of the human genome constitutes genes that code for proteins, several…

The EMSA – Teaching an Old Dog New Tricks

The EMSA – Teaching an Old Dog New Tricks

Probing Nucleic Acid-Protein Interactions with EMSA The electrophoretic mobility shift assay (EMSA) is a powerful technique for detecting specific-binding of nucleic acid-protein complexes. Over the past 30 years, EMSA has been the “go to assay” to investigate the qualitative interactions between nucleic acids (DNA or RNA) and nucleic-acid binding proteins. Through the use of radio-labeled…

RNAseq Library Preparation: From Cells to cDNA

RNAseq Library Preparation: From Cells to cDNA

RNAseq libraries, also called whole transcriptome shotgun sequencing libraries, provide a snapshot of cellular processes. This allows the researcher to gain information regarding changes in transcriptome in response to environmental changes, during disease, or after a drug application. RNAseq libraries also allow for the detection of mRNA splicing variants and SNPs. RNAseq libraries have virtually…

Picking the Right DNA Isolation Kit for Your Application

Picking the Right DNA Isolation Kit for Your Application

If you plan to work with purified DNA in the lab, it’s likely that you will use a commercial DNA extraction kit to isolate and purify your DNA of interest. With so many types of kits available, it can be a major challenge to choose the best one to use when working with an unfamiliar…

Are You In(to) Situ? – Putting Together Your First RNAscope® Assay

Are You In(to) Situ? – Putting Together Your First RNAscope® Assay

You are thinking of trying out RNAscope®. After all, RNAscope® holds promise for increasing the sensitivity and specificity of your in situ hybridization. Yet, getting started can be a little overwhelming with the numerous kits and reagents available in the RNAscope product line. Here’s an overview of your options to help you navigate to the…

RNA Strandedness: A Road Travelled In Both Directions
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RNA Strandedness: A Road Travelled In Both Directions

For most molecular biology purposes, DNA is thought of as a string of nucleotides running from 3’ to 5’, and the corresponding mRNA sequence is complementary to this DNA string. However, visualizing this quirky DNA structure for what it is – two antiparallel strands joined together – it quite important for many applications, such as…

The Next Big Thing: Alternative Polyadenylation
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The Next Big Thing: Alternative Polyadenylation

What Is Alternative Polyadenylation? Processing of mRNA and its regulation plays a fundamental role in gene expression. As science progresses, alternative polyadenylation takes center stage in the undercurrents of gene expression. 1,2 Polyadenylation is part of the pre-mRNA maturation process and involves polyadenylation of the 3’ end of the emerging RNA.  This process happens to…

The Importance of Non-coding RNAs
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The Importance of Non-coding RNAs

What Are Non-Coding RNAs? What was once considered “junk” may end up being the most important part of our genome. Non-coding RNA (ncRNA) is RNA that is transcribed from DNA but diverts from the “central dogma” because it does not code for proteins. NcRNAs are ubiquitous in eukaryotes: while 90 percent of eukaryotic genomes are…

Protocols for Cloning Without Restriction Enzymes or Ligases
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Protocols for Cloning Without Restriction Enzymes or Ligases

There are many cloning methods that do not require restriction enzymes or ligases. Read below to learn about how to achieve seamless cloning results via Topoisomerase cloning, SLIC, and Gibson. Method #1: Topoisomerase Technology Topoisomerase technology requires no special primers and no ligases – it is as easy as cloning comes. This technology is based…

Cloning Methods: 5 Different Ways to Assemble

Cloning Methods: 5 Different Ways to Assemble

Over the past few decades molecular biologists have developed procedures to simplify and standardize cloning processes, allowing vast arrays of artificial DNA structures to be more easily assembled. Are you familiar with all the cloning options out there? Let’s look at five different cloning methods you can use to get your construct. At the end…

Ligation Independent Cloning Primer Design
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Ligation Independent Cloning Primer Design

Ligation independent cloning (LIC) is an easy and effective method to ensure successful cloning, all without the need for ligation. As easy as the technique is, designing primers can be a bit tricky. In this article, we will present a quick overview on primer design for ligation independent cloning.

A Beginner’s Guide to Lentiviral Transduction

A Beginner’s Guide to Lentiviral Transduction

The use of viral delivery systems to transduce cells for gene and protein investigations has become prominent over the last 20 years. In particular, the use of lentiviral vectors permits stable expression of your gene of interest. This is all possible with a little bit of nucleic acid magic. Lentiviruses (a genus of retrovirus) express reverse…

Old Reliable: Two-Step Allelic Exchange

Old Reliable: Two-Step Allelic Exchange

Manipulating the genes of organisms is crucial for studying their functions. In times before genetic engineering, scientists would shoot bacteria with X-rays or expose them to destructive chemicals until spontaneous mutations would arise. Fortunately, current methods are more sophisticated and less torturous. Researchers now use more directed techniques to introduce mutations. There are several ways…

RNA Isolation from Drosophila – Don’t Let the Cuticle Scare You!

RNA Isolation from Drosophila – Don’t Let the Cuticle Scare You!

Isolating RNA from either Drosophila larvae or adult heads can seem a bit daunting, primarily due to the presence of the cuticle. The cuticle is a protective exoskeleton comprised of insoluble collagens, cuticlins, glycoproteins, and lipids. While it may take some force to remove the cuticle, you can do this easily and without compromising your…

Small Differences that Matter: Detecting Microsatellite Polymorphisms

Small Differences that Matter: Detecting Microsatellite Polymorphisms

If you have any training in genetics, chances are that during the course of your education you ran into those funny little sequences called microsatellites. These are repeated tandem motifs 1-6 nucleotides long, scattered all over our genomes. These used to be called “junk DNA,” because researchers thought that the repeats served no purpose. Nowadays,…

DNA Footprinting

DNA Footprinting

Studying DNA–protein interactions is an important aspect of molecular biology. Researches use a variety of methods to study these like the chromatin immunoprecipitation (ChIP) assay, electrophoretic mobility shift assay (EMSA), DNA pull down assay, luciferase reporter assay, filter binding assay, yeast one hybrid system, etc. Another interesting assay that helps investigate DNA–protein interactions is the…

Bacterial Transformation Troubleshooting for Beginners

Bacterial Transformation Troubleshooting for Beginners

The first time I did a transformation was when I worked with site directed mutagenesis. I cloned a protein sequence into the p15TVL vector, created my mutants (but that’s another story), and was finally ready for the next step: transformation and expression of my desired protein. Little did I know that my enthusiasm would fall…

Restriction Enzymes: Five Things to Consider Before you Chop!

The use of restriction enzymes to characterize DNA has been popular since the 1970s. Today, this “old school” technique is still one of the easiest and fastest ways to assess DNA sequences. Like most lab reagents, restriction enzymes can be fickle and you should bear a few things in mind when using them. Generally, sticky-ended enzymes have greater…

Those Site-Specific Recombinases in Your Tool Kit

Most of us are aware of genetic engineering systems like Cre-Lox, TALENs, Zinc finger systems, and of course, CRISPR-Cas9.  These are all examples of CSSR- Conservative Site-Specific Recombination. We use these site specific recombinases routinely, but do we really know about them or what the future hold for these tools? It turns out that CSSR…

Using Synthetic DNA For Long Term Data Storage

The amount of data requiring long-term storage is growing and accelerating. Current long-term digital storage technology cannot keep up. Imagine roughly 2.5 QUINTILLION bytes of data being created everyday in this world1–2 as more computers and network infrastructure come online. For average users, a long-term storage solution is probably not an issue. However, organizations and…

FISHing for miRNAs in Archived Tissues? Yes, It Is Possible!

We use fluorescent in situ hybridization (FISH) techniques routinely to detect DNA or RNA sequences in tissues, but what about micro RNAs (miRNAs)? No worries, FISH is now optimized to meet the challenge. To help you get going with the method, here’s what you need to know. The first thing that comes to mind when…

Get Your Polymerase Cycling Assembly Oligos Together

Get Your Polymerase Cycling Assembly Oligos Together

The polymerase chain reaction (PCR) is the backbone of many lab techniques. In short, it allows for the exponential amplification of a specific segment of DNA. Through the use of primers encoding restriction enzyme sites, these amplified fragments are used in downstream cloning procedures, usually leading to the insertion of one, maybe two, PCR fragments…

How to Perform DNA Extraction from Dried Blood Spots Using Chelex Resin

Every bio- scientist who wants to analyze DNA knows that the process begins with the extraction of DNA from cells of interest. These cells could be RBCs, parasites, or bacteria to name a few. Furthermore, there are various DNA extraction methods1  to choose from depending on sample type, downstream analysis, and so forth. Many scientists…

Benzyl Isoamyl Alcohol: a Novel, Bizarre, and Effective DNA Purification Tool

DNA Purification We all use our favorite techniques for DNA cloning, such as Gibson assembly, TOPO cloning, ligation independent cloning (LIC), and TA cloning. However, DNA purification methods themselves, haven’t changed all that much since the 90’s. Historically, the introduction of phenol extraction in 1956, to purify nucleic acids from rat liver, rapidly replaced previous…

Tips and Tricks to Get Around Low Plasmid Yields in Agrobacterium tumefaciens

Tips and Tricks to Get Around Low Plasmid Yields in Agrobacterium tumefaciens

A while back, one of our readers asked for a quick and easy and quick way to extract plasmids from transformed Agrobacterium tumefaciens cells. They pointed out that plasmid copy number is often low in Agrobacterium and that yield can be poor in alkaline base miniprep protocols. The short answer is that there is no…

Quantifying & Assessing RNA: TE or not TE?

Quantifying & Assessing RNA: TE or not TE?

Red Pill or Blue? Carrying out science often involves many difficult decisions! I see it all the time in RNA protocols – the “gracious” option of using purified water or Tris-EDTA (TE) buffer to dissolve (or elute, if you are using column purification) RNA. When I was trained in assessing RNA using UV spectrophotometry, graduate…

Benefits of the GUS Gene Reporter System in Plants

Gene reporters enable valuable insight into gene expression. The GUS gene reporter system is one of the popular and common plant reporter systems. GUS,  is short for glucuronidase, an enzyme in the bacterium E. coli. GUS is a good reporter for plants, as it does not occur naturally, and thus, has a low background. With…