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Dear Aunt Yersinia, I work with a yeast vector where my gene is under a Gal promoter. My boss told me to grow yeast in medium with sucrose, and then add galactose to induce the promoter. I did it, but my protein is not induced; the Western blot is empty. I sequenced a part of…
If you ever used a site-directed mutagenesis kit or ligation-independent cloning, then you also used restriction enzyme Dpn I. But what does it do and more interestingly, why? Restriction enzymes and methylases: the yin and yang of bacteria Usual restriction enzymes, the toolkit of genetic engineering, are one half of the “yin and yang” pair…
The wide range of applications of PCR has led to an ever-growing list of variants of the technique. While some are optimizations to suit specific requirements and are very similar to basic PCR, others completely turn the technique on its head to formulate novel creative applications in various fields. This article lists some variants of…
Scientific conferences are a peculiar throwback to XIX century, when bearded white men in wool suits were meeting in ale houses to discuss the latest experiment they did at home – once. In our time of instant communication and telepresence, conferences are obsolete and I’ll tell you why. 1. The main reason not to go…
Two different sensors are generally used in cameras for microscopy: Charge Coupled Devices (CCD) or Complementary Metal Oxide Semiconductors (CMOS or sCMOS). Although there are a number of similarities between the two sensors, differences in the way they function can have an effect on image capture time as well as signal-to-noise ratio. Let’s take a…
Cytokines, those small proteins that modulate immune cell responses, once translated are normally secreted rapidly out of the cell. So, previously we could only check the levels of cytokines secreted in the supernatant, but we wouldn’t know which cell was producing which cytokine. But what if we had a way to keep the cytokines inside the cell? Then we…
Your lab is full of non-chemical hazards that can explode, stab, kill, and – as if that wasn’t enough – bite. Here’s a list of those hazards to remind you why Environmental Health & Safety exists! 1. Centrifuges Centrifuges are dangerous, especially when not cared for! An unmaintained ultracentrifuge imploded in an American lab in…
You’re about to start that big project you’ve been dreaming of for years. You’ve identified a potential miracle compound and want to figure out how it affects gene expression. But how are you going to do it: with next gen sequencing or a microarray? Especially if you are new to this area of research, the…
Like the legendary fight between boxers Bowen and Burke in 1893, the cell cycle in some cells goes on and on..round 1, round 2, round 3, round 4…before the final bell is rung. Nucleotide analogs, like BrdU or EdU, are great for examining 1-2 cell cycle division(s). In many studies though, for example the proliferation…
Westerns can be tricky and time-consuming, so make the most of your precious membranes and their proteins. Learn how to properly strip off your antibodies and re-probe with another primary antibody. Why you should strip Scientific reasons: To conserve protein samples that are limited or expensive. So that you can analyse the same sample with…
There is a right way and a wrong way to set up a PCR laboratory. Because of PCR’s tremendous ability to amplify small quantities of DNA/RNA template, even the smallest of template contamination can become a huge problem in PCR. However, contamination does not have to be a problem in your laboratory. Read below to…
The thing that was most difficult for me as an R beginner was plotting graphs with error bars – there is no concise way to do this with base graphics. There are workarounds, often using the ‘arrows’ command, but isn’t there a simpler way? Yes, in fact there are a handful of plotting packages for…
More than a pun on the explosive growth of sequencing data, BLAST makes annotation and comparisons of similar sequences much easier. Created by a group at the U.S. National Center for Biotechnology Information in 1991, the Basic Local Alignment Search Tool is arguably the most heavily used tool for sequence analysis (that’s available for free,…
Good news lab workers! Always hated the tedious work of designing a cloning strategy? Or maybe always dreamed of pooling all the reactions in one tube, just to save time? Thanks to Mother Nature, and her wonderful type IIS endonucleases, this is now possible! What is this wonderful enzyme? Type II enzymes are one of…
Most of us hate cleaning and are often hard pressed to find time to clean our homes, never mind our laboratory space. However, an annual spring clean and maintenance of a regular cleaning rota/regime will contribute to an efficient, organized and harmonious lab environment. This is increasingly important in communal lab spaces where multiple staff…
Biosafety is one of those things many scientists don’t take seriously. I would guess, that like politics, there are 40% who believe biosafety is ‘over-emphasized’ and 40% who swear by biosafety. 20% are undecided. Needless to say, I’m on the side of biosafety. And here’s why: “CDC announced today that approximately 75 Atlanta-based staff are…
You have your favorite protein in mind and are ready to set up some exciting experiments to show what it does and how it does it, when it is active, what other proteins it modifies, and how it affects your cells. There is one slight problem. You need to fish your favorite protein out from the…
Let’s imagine the following scenario: You are researching a biosynthetic pathway in your favorite fungus. You know that this pathway produces a family of toxic compounds, and you want to see if you can block this pathway (or parts of it) with an antifungal drug. You have a control (no antifungal) and samples that have…
Flow cytometer and cell sorter manufacturers have invested considerable resources to design instruments that are the “fastest in the ‘hood” either in terms of cells analyzed per second, or in total throughput. The general idea is the faster you can go, the quicker you can identify rare cells, and produce sorted populations containing large numbers…
If you’re an HPLC guru, then you probably think that everyone should be using HPLC. And you might have a point – HPLC is very powerful and has broad applications across many fields. But it isn’t the answer to every problem. HPLC (high-performance liquid chromatography) is used to separate mixtures of compounds based on their…
As frustration goes, cloning is often up there with trying to thread a camel through the eye of a needle. You do everything carefully: prepare your vector and fragment DNA, cut them as the restriction enzyme manufacturer instructs (complete digest in five minutes!), ligate, transform and go home in anticipation of a good number of…
PCR (Polymerase Chain Reaction) is a biochemical technique developed by Kary Mullis in 1983 that is used to create large quantities of a sequence of DNA. Since this method of mass-producing DNA was first introduced, it has become significantly less labour intensive, more economical, and more routine. The technique relies on a few key players…
My mom is a microbiologist and so I was a lot more informed about bugs than most kids. In fact, I probably gave more than one classmate nightmares with my talk of there being 10 times more bacteria that make up the human body than human cells. I remember my mom working by a Bunsen,…
While the classic approach to molecular cloning – using restriction enzymes to excise a DNA fragment of interest – is as useful as ever, new techniques that make cloning faster, easier and more versatile are available. As a smart molecular biologist, you should be examining each of them to see whether or not adding them…
After a very naïve start in flow cytometry thinking that published protocols will work without fault – needless to say, that did not work out in my favor – I realize now that following a few simple steps can go a long way, and will undoubtedly save you time in the end. So, here are…
After you finish immunoprecipitating a protein or purifying a subcellular compartment, you need to identify what proteins you purified. You could attempt to identify your purified proteins the old fashioned (and slow!) way by running a multitude of Western blot. But rarely do labs have unlimited funds for Western blot antibodies. And lets face it,…
If you work with an HPLC, then you know the frustration of going to use the machine and finding it in disarray. If you’re new to using an HPLC, then the machine can be intimidating to use and you might not know the ins and outs of using it. Here’s an article that has a…
Like yin and yang, apoptosis has a duality. While it is is a pathway used in the normal maintenance and development of tissues in healthy organisms it also had a dark side. As you can imagine, apoptosis is a tightly regulated process – controlled by the integration of multiple pro- and anti-apoptotic signals. Ultimately the induction…
Your advisor tells you that he wants you to use HPLC to analyze your compound. You know that you’ve heard of this technique before, but you can’t remember what HPLC stands for, let alone how to go about doing it! We’ve all been there, though. Fear not! In this article, we will remind you about…
Your advisor tells you that he wants you to use HPLC to analyze your compound. You know you’ve heard of this technique before, but you can’t remember what HPLC stands for, let alone how to go about doing it! We’ve all been there, and I bet you wish you had paid more attention in that…
They say a magician is never supposed to tell the secret behind his tricks. It’s a good thing then that I am a molecular biologist and not an illusionist. Because once I learn a good trick, I feel the need to spread it like dandelion seeds floating on a breeze. That’s what inspired me to…
The Scene of the Crime The body of a woman is found behind an abandoned warehouse on the outskirts of town. Forensic experts carry out a technical examination of the scene and suggest strangulation as the cause of death. The absence of bloody footprints or weapons means there are no obvious leads to the killer….
It seems like every movie that needs a shot of a scientist doing their sciencey-thing either gets the person to pour one pretty liquid from one flask into another or to stare intensely at a test tube with a look of knowing so much more than you ever could. Another favorite shot is of someone…
Blocking is the essential third wheel in any antibody/antigen relationship. Correct blocking buffer can perfect your antibody’s ability to bind its antigen, while bad blocking can make specific antibody binding near impossible. Don’t let bad blocking be a stumbling block in your Western blot experiments – read on to find out what blocking achieves and…
What is one of the first things you do when you sit down at the flow cytometer and start looking at your cells? You start drawing polygons and setting gates. To the neophyte the gating process can look a little random – why do you exclude those dots but not these? But gating in flow…
We’ve been slowly coaxing you along in our R tutorials. We’ve introduced what R is, gave you a basic tutorial into how to use R and also spent some time learning how to explore your data with R. By now you are probably itching to use R for more complicated analyses. To indulge you, I…
Like athletes running on turf versus sand, the gel you run your DNA through can highly affect your results. The two main types of gels that people use for DNA electrophoresis are agarose and polyacrylamide (PA) gels, but figuring out the differences can be confusing. Basically, you choose a gel based on two main factors:…
Here are a few handy hints for dealing with your flow cytometry core facility colleagues. They are an amazing resource and are the people with the power to get experiments done.
Grad school is a big investment of your time, with a lot riding on a successful relationship with your mentor. Unfortunately, you may have realized that the relationship is not working and resists improvement. You’ve taken the steps to switch to a new mentor. Now comes the hardest part. What do you actually say to…
Run to red! It’s a mantra I learned when first using gel electrophoresis to separate DNA molecules. This can save you a lot of frustration and humiliation in the lab (stage right: a complaining scientist who swears the equipment is broken as a supervisor facepalms in embarrassment). But what about how does this jell-o like…
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