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Over the years, I’ve had my fair share of ups and downs in the lab. The latter quite often centering on a failed or plainly weird PCR experiment. As I’ve gone on and become ever more fastidious about my lab practices I’ve realized that the majority of these little calamities were perfectly avoidable. In my…
Every protein is unique and thus every protein has its own set of production and purification challenges – many of which cannot be predicted. Therefore to successfully produce and purify your favorite protein you need to know and understand these five unpredictable protocol variables (X factors). Tweaking these X factors just might be the difference…
Colocalization blues (and reds and greens) Trying to find if and where two epitopes co-localize (or, to be more precise, where they are found in close proximity) may seem easy at first: 1) Bind your two epitopes with primary antibodies from two different species, 2) bind these primary antibodies with two secondary fluorescent antibodies, one…
A marriage of sorts Fluorescence resonance energy transfer, or FRET, is often done using a microscope, which means it can be difficult to analyze large numbers of cells in one sitting. One way to overcome this, is by combining FRET with fluorescent-activated cell sorting (FACS), giving you a high-throughput method to screen for protein interactions…
Yup, we really are in the digital age…even the pathology is digital. Facebook, Instagram, Tumblr. It has never been easier to take pictures and share them. The digital revolution is upon us and nothing is safe, even your pathology samples. Digitizing your pathology samples can help you better organize and manage pathology for later. And…
What do bunnies, coins and PCR have in common? They all have tails! (ha ha!) Thermal asymmetric interlaced PCR or TAIL-PCR is used to sequence and analyse unknown DNA fragments that are adjacent to known sequences. Think of it as being rather like networking. You know you want to get to know someone so you…
As researchers, we are constantly on the lookout for new and improved ways to analyze, detect and quantify our favorite protein or gene. Luckily, we don’t always need to reinvent the wheel! PCR-ELISA is a good example of where two commonly used techniques have been merged together to create a very powerful analytical tool. What…
When it comes to Western blotting, there’s no denying it: Your membrane is a key player. After all it is the physical scaffold that holds your precious samples and it needs to be up to the challenges you throw at it. But depending on your protein’s properties and your downstream detection steps, finding the optimal…
Single-molecule localization techniques are another way to achieve super resolution. Discover how the different variants work, and get info on what researchers are doing with them.
The golden rule of flow cytometry, especially cell sorting is: ‘Put good cells in and get good cells out’. When you sort you might not get good cells out and you may not get the numbers you were expecting. In my previous article I touched on a few reasons why your cell numbers might be…
Consider a jigsaw puzzle. While most of the pieces have a different picture on their surface, all pieces fit together in an interlocking pattern. As unlikely as it may seem, restriction enzymes from different organisms can produce interlocking pieces of DNA – so called compatible cohesive ends (CCE). These are pieces of DNA, which fit…
We all know, generally from bitter experience, that experiments don’t always work first time and that sometimes the little things that govern success are the things that get left out of that online protocol! So whether you are assessing proliferation by nucleotide incorporation or by dye dilution, here are some handy hints to help you…
You walk into your tissue culture room to find a window open, the incubator’s humidity tray empty and a pipette lying on its side in the hood on a used glove… After you have found and torn to shreds the person responsible for this monstrous act (!!), consider posting the below tips to help those…
Look Ma, No Kit! Unlike with kits, there are no propriety reagents in a DIY protocol. You know what exactly is in each reagent. The DIY approach is catching on!
You are finished with your super-arduous experiment. Now the fun part! To “read” your results with a microplate reader, but do you know which microplate reader to use? Or even that there are different types? Have no fear. I am here to help ye, scientific one! Read on for my pointers on picking the right…
If there are a million cells of interest in your sample and you pass them through a sorter, you might expect to get a million cells back. But you don’t – and here is why. Hardware aborts A hardware abort occurs when an instrument can’t process the information about an event because it is still…
Glucose is the preferred food source for E. coli, however when glucose levels drop, E. coli need to look for other ways to feed themselves. One way in which they accomplish this is to replace glucose metabolism with lactose metabolism. The induction and control of lactose metabolism is complicated and its process has been exploited…
The completion of the Human Genome Project in 2003 ushered in a new era of rapid, affordable, and accurate genome analysis—called Next Generation Sequencing (NGS). NGS builds upon “first generation sequencing” technologies to yield accurate and cost-effective sequencing results. Fred Sanger sequenced the first whole DNA genome, the virus phage ?X174, in 1977. In that…
Antibodies are one of the most important tools in molecular biology. Basic researchers use antibodies to identify, locate, isolate and quantify specific proteins. Clinical researchers use antibodies to target a specific drug (such as a chemotherapeutic agent) to a particular cell. Because of the cell specificity provided by the antibody, a higher amount of antibody…
The magnification and viewing of samples using a microscope relies on both the objectives and the eyepieces working harmoniously together. If you buy a ready-to-use microscope, then the objectives and the eyepieces which are fitted as standard will be designed to complement each other. On the other hand, if you are designing and building a…
If I piqued your interest in the first post about my new e-Book ‘The Bitesize Bio Guide to Protein Expression – a Bitesize Bio eBook’ check out this excerpt from the book explaining what an expression system is and how to choose the right one. What is an expression system anyway? There was a time…
An operon is a functioning unit of genomic DNA that contains a group of genes controlled by a single promoter. Put simply, these genes share information needed to create the tools for a particular task so they share a promoter ensuring they’ll all be transcribed together. The lac, or lactose, operon is found in E….
As with some of the greatest discoveries in science, from penicillin to microwave ovens and play-doh, PCR was discovered serendipitously. Thanks to the work of many scientists, including Watson and Crick, Kornberg, Khorana, Klenow, Kleppe (so many K’s…) and Sanger, all the main ingredients for PCR had been described by 1980. Like butter, flour, eggs,…
Cell sorters do not operate by magic, even it looks that way. It’s about the application of physics, electronics, fast computers and formation of droplets. Whether you bring your cells to a flow core to be sorted or you sort them yourself, it important to know how the cell sorter works. Cells can be sorted…
A promising study on using gene expression to develop personalized treatments for ovarian cancer. A report of surprisingly high levels of differential gene expression among different ethnic groups. The announcement of previously unsuspected levels of physiological diversity in Plasmodium falciparum, the parasite that causes the most deadly form of malaria. What do these three seemingly…
If efficient cloning is what you are after, you must give Overlap Extension PCR Cloning a go! This restriction enzyme and T4 DNA ligase-free technique is faster, more reliable and easier to troubleshoot than traditional restriction methods. With only two PCR reactions required, you can insert a DNA fragment into a plasmid without spending time…
Super resolution microscopy shattered the lower resolution limit offered by traditional confocal microscopy, enabling us to see more detail. Discover the different super resolution techniques, how they work, and what they are being used for.
It happens to the best of us. You’re minding your own business and suddenly out of nowhere your mastermix is a bubble bath and your primers are enjoying a froth party. Let’s talk about how to deal with these foamy fiends! When you’re making up your mastermix, you could have a variety of ingredients going…
“What Have You Done To My Cells??!!!” This cry of pain from researchers, frequently aimed at core facility operators, is heard after receiving incomprehensible data for an invaluable tube of cells. Equally baffling to the trained user of flow cytometric instrumentation is when data emerges that is either unreliable or inconsistent with the known properties…
MIQE what’s that? When writing dPCR materials and methods for a paper have you ever pondered what information you should include? This is where the MIQE guidelines will really help. Guidelines for minimum information required for publication of a digital PCR (dPCR) experiment were published by JF Huggett et al. in 2013. These were a…
Ever done a multicolour flow cytometry experiment, run all your controls, done your compensation and then started to analyse your data and realised that you can’t work out where to put your gates? To err is human… When acquiring data on a cytometer, there can be measurement errors due to counting statistics, errors in processing…
It has been said that “Incomprehensible jargon is the hallmark of a profession” (Kingman Brewster, Jr) and while I cannot speak for other professions, as a biologist I am inclined to believe it. So whether you need to cover your qualifying exam bases, want to avoid looking like an idiot to your coworkers, or need…
No, this is not a call for Geeks to take over the world – just a tiny part of it – the part that ensures success in all experiments or at least a good way to analyze them if they fail. There are all too many entry points for error and variability that can be…
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…
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…
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…
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