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Some viral vectors are the little black dresses of cloning and expression experiments: They work for almost any occasion and always give you the results you were hoping for. Other vectors are more like ballgowns that only come out of storage for special occasions. Let’s wade through all the information out there and take a…
It doesn’t matter how excited you are about the research or how intriguing the biology is, if you cannot record it – and record it well – it won’t matter. Here I will tell you how to take the perfect electron micrograph.
The word ‘retrovirus’ evokes images of HIV, the AIDS pandemic and a desperate worldwide effort to defeat this mighty adversary. But on the flip side, scientific research has managed to tame the virus and use it as a tool for advancement. Retroviruses are commonly used to introduce genes into mammalian cells to express or knockdown…
You had been happily measuring your DNA samples with your NanoDrop for a while, until… you noticed that your results are not reproducible *gulp*.
Read on – here are 10 points that will help you troubleshoot the issue.
Alu sequences are repetitive DNA sequences that are widely dispersed within the human genome. These “junk DNAs” are not as useless as one might think. An interesting method to use them is to quantify the number of integrated Human Immunodeficiency Virus (HIV) genome copies using Alu-PCR.
The world as we know it couldn’t exist without microbes. It is estimated that there are tens of trillions of bacteria living just in and on humans. (Ewww?) One trillion has 12 zeros. To put that in perspective, the entire human population on earth is only 7.3 billion with a measly 9 zeros. With that many microbes…
Wow, you’ve done it! Your experiment worked and your boss asked you to write it up for publication in your favorite journal. Where to start with presenting your flow data? Take a deep breath, help is in hand in the form of the MIFloCyt Guidelines. As with other scientific techniques, there are ‘Minimum Information about…
Now you’ve got great sequencing results, thanks to Nick’s article on improving sequencing results. Now what? Well now you need some software (preferably free) to analyze your data. BioEdit is a good option. But what I have to offer today is a much lighter and equally handy tool. It’s called Artemis and was developed by…
How to Obtain a Purer PCR Product and Reduce Non-specific Amplification Unless you’ve gotten your hands on some miraculously specific primers, amplification of only your target sequence without non-specific amplification can be very challenging. Thankfully, a clever and surprisingly simple solution is at hand! A Quick Recap of the Basics In PCR, you design your…
What is a luciferase assay and what is it useful for? A luciferase assay takes advantage of the innate bioluminescent properties some organisms exhibit, most notably the firefly. The firefly can convert luciferin to oxyluciferin in the presence of the enzyme luciferase to emit light. The most common scientific assays utilizing luciferase are reporter assays…
Retroviral transduction is becoming a popular choice for gene delivery into mammalian cells and has multiple advantages over other techniques. If you decide to start work on this useful technique, here is how you can go about it: Step 0: Obtain permission First and foremost, do you have the permission, authorization, and training to work…
Take a look at the dotplot below, are you happy with the way it’s presented? Do you think that you could recreate that experiment? If you were a reviewer, would you accept that figure? Sure, it’s flow plot, it shows 3 populations of which two are gated. Read many journals and you will see data…
You open the incubator in the morning and to your dismay there are a hundred glorious colonies… on your vector-only control plate. While there are a number of potential causes, I’ll highlight a few of the more likely culprits and their solutions.
Have you ever wondered what a cell’s life is like? Do they constantly communicate to each other or do they just go on their own daily business? There is an easier way and it involves super advanced molecular “crayon” technology.
I’ve never run a sequencing gel in my life, but people around me did, and they spent a lot of time on getting it just right. Although the principle described by Sanger in 1975 sounds straightforward (1), sequencing gels are very long and very thin – less than a millimeter thick! They were easy to…
Even in the most basic applications, fluorescence microscopy can be a very powerful technique. Simply put, the ability to actually see the biology you are interested in cannot be matched in directness. Often, the aim of fluorescence microscopy is to observe the effect of an experimental manipulation. Ultimately, you would like to know that the…
In part I, I answered the question, “How do proteins in thermophiles survive under high temperatures?“ In this part, I’ll look look at how nucleic acids survive -thrive, even- in conditions that are too hot for most of us, but ideal for a number of organisms, including the one that gave us Taq polymerase and…
Genomics, transcriptomics, proteomics, metabolomics – words that in 2015 sound very familiar even to a freshman in any biology field. Although most have heard those words before, I keep encountering students or even post-graduates who find it difficult to explain what they are. So, to make things easier here is a peek behind the curtains…
As biochemists, we routinely run SDS-PAGE to analyze our proteins. Imagine the time and effort you are going to save when you can run every gel to perfection.
The concepts of brightness and contrast are so general, and the issues related to them so many, that it may seem strange to have a single brief article with such a title. Indeed, when we speak about brightness, we can think about the brightness of the light source, the aperture and magnification of the lens,…
Western blotting uses electrophoresis and antibody-epitope affinity to give a semi-quantitative and (theoretically) clear measure of protein abundance. It’s a long procedure, filled with many steps—and even more room for error. Learning to troubleshoot certain problems is incredibly important for continued success with this technique. So what do you do when your final imaged product…
When we are doing a double labelling, it makes good sense to choose two fluorophores with spectra widely spaced apart to avoid mixing. Why combine GFP with Rhodamine, when you can combine it with Texas Red?
Phenol-chloroform extractions and ethanol precipitations can be a royal pain, but here are some tips to help you get tip-top results!
If you’re planning on using lentivirus for your next experiment, chances are you’re wondering how much virus to use. For in vitro work, multiplicity of infection (MOI) is the theoretical number of virus particles applied per target cell. That is to say, if you have 1 million cells and you want an MOI of 5,…
Recently, I have witnessed the uprising of various next generation sequencing (NGS) platforms and it’s quite interesting because each platform uses a different method. Previously, I’ve written about the exciting possibility of nanopore sequencing—a new sequencing technology based on the “signature” electrical currents generated as a single strand of DNA passes through the nanopore. The…
So, you’re sitting there with your list of significant SNPs, thinking, “what do I do now”? Hopefully this article can point you in the right direction! So far, you will have extracted genomic DNA from your organism of interest, sourced the SNP chips required, and had the DNA run on these chips. The chips will…
While overexpressing a gene of interest can provide a look into its role in a cell, sometimes it is necessary to control the expression of a gene. You may want to dictate the timing of the protein’s expression or lower its expression level to adequately understand its function. This is particularly relevant when studying genes that…
Variability is the Achilles’ heel of research. It can often confound our results and lead us astray searching for solutions. There are two kinds of variability, the first is biological variability. This represents the stochastic nature of the sample you are working with and the inherent differences between samples from the same conditions. There is…
If you’re hoping to reel in a positive interaction between a protein and an RNA sequence, try to catch a winner with a yeast three-hybrid assay. What is yeast three-hybrid (Y3H)? The Y3H system is based on the same principle as a yeast two-hybrid– namely, that the DNA binding domain and the transcription activation domain…
Flow cytometry is a fluorescence-based technology, as is fluorescence microscopy and confocal microscopy. Fluorescence is fundamental to how a cytometer gathers data, but I am often surprised, as a core manager, at how little new users know about the process of fluorescence. So, this is where I always start the training process. Let’s get physical…
Welcome to the last article in this series! Last, but by no means least, we will look at the importance of plant miRNAs and how they differ from their animal counterparts. When/How Were Plant miRNAs Discovered? Plant miRNAs were first described in 2002, a decade after the seminal miRNA study in the nematode C. elegans…
Digital PCR (dPCR) is a next generation qPCR that you might just need for quantitation and comparison of minute genetic copy differences.
Three dimensional cell culture mimics the extracellular matrix (ECM) that offers the structure and support for cells in vivo, thus creating the complex architecture and network required for cellular communication. For 3D cell culture beginners (or enthusiasts), the information available may seem overwhelming. It sure was for me. But it can be simplified. For example,…
It’s happened to us all, you are ready to run your samples on the cytometer and you can’t see your cells on the screen. Here are a few tricks to troubleshooting this: Cytometer vs. computer connection The different types of cytometers will need different orders for switching on the cytometer and computer. Some are cytometer…
Here’s a tip that you may find useful if you are expressing proteins in E.coli using a lac promoter-based expression system, e.g. pET, in LB medium (L-broth). Lac expression systems are typically induced in the lab using IPTG (isopropyl-beta-D-thiogalacto- pyranoside), which is a non- hydrolyzable analog of lactose, the natural inducer of the lac operon….
Is your goal to purify a substantial amount of a specific protein? Do you have a quantity of a molecule that binds your protein of interest? If so, generating a custom affinity matrix may be just the trick you need to purify your protein of interest by affinity chromatography. Customizing your affinity chromatography is an…
The most comprehensive way to evaluate DNA concentration and purity is to use both UV spectrophotometeric measurements and agarose gel eletrophoresis. This quick reference guide gives an overview of the information that can be derived from both. UV spectrophotometric measurement of DNA concentration and purity DNA itself, and most of the common contaminants found in…
Flow Cytometry is a great way of seeing how many of your cells express a particular marker and how much of it is there. We do this by measuring fluorescence, but, as with all measuring systems, there will be signal that we are always trying to measure the above the noise. The signal that we…
Imagine trying to build a house without power tools: It’s completely doable – after all, people did it that way for centuries – but it’ll take you a lot longer and has limits. Similar to this, modern day biology now has its own set of “power tools.” So while you could do biology the old-fashioned…
People would have said that a USB sequencer not much bigger than a memory stick which could sequence genomes in 50kb+ read-lengths was impossible “’Star Trek’ technology!” Now, that futuristic technology is here.
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