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Every PCR battle is the same: Too little amplification of your target DNA versus too much amplification of off-target DNA. But you can win the PCR battle and amaze your co-workers by mastering the use of PCR additives. PCR additives usually work one of two ways: 1) By reducing secondary DNA structures and thus increasing…
“A two photon microscope has higher sensitivity than a normal confocal microscope, because it uses two photos instead of one!” Yes, I can bear witness that this phrase has actually been uttered, and it was not by an undergraduate student. No exception to the rule The condensation of various levels of misunderstandings in this statement…
Ever had a blot so bad it looked like a Rorschach test? We have ten things that might be going wrong with your western blots and how to fix them.
DNA sequencing (PCR, Sanger or next-generation sequencing (NGS)) is a now familiar part of any molecular biology lab. But ‘RNA-seq’, the so-called “Cinderella of genetics”, is now becoming the belle of the ball, providing new insights into this most central molecule of the ‘central dogma’. The many flavors of RNA Whilst genomic DNA is the…
If you remember from one of my previous articles (if not, you can read it here!), we introduced ‘fluorophores’. These are basically substances (natural or synthetic) which have the ability to absorb light at a low wavelength and re-emit at a higher wavelength. In other words- they fluoresce! In this article, I’ll introduce the three…
Buffers are often taken for granted, but they can make or break an experiment. In previous posts, we’ve talked about the wide ranges of buffers available for biological research and the characteristics of a “Good” buffer. Organic buffers are not inert! They can interact with your experimental molecule, or change pH due to changes in…
As widely used as it is, isolating RNA remains one of the more finicky protocols. Just about anyone who has performed the technique has their own personal tips and tricks to successfully isolate intact RNA from their samples with consistency. Although RNA can be somewhat unpredictable since it is so labile, there are a few…
The issue of mouse-on-mouse background is only a cause for concern for the histotechnologist working within a research environment. Those working in a diagnostic setting will probably never experience this as they will be working with human tissue with antibodies raised in a variety of species- but one species that won’t be used is human!…
Do you wonder if your favorite protein interacts with another protein? Do you wish that you could shine a spotlight on your protein to determine its binding partner? You can use co-immunoprecipitation (Co-IP) to find your protein’s partner. This article will get you ready for your first Co-IP, provide a handy Co-IP protocol, and discuss…
You may already use fluorescence as a tool in your microscopy and imaging work, but, do you know exactly what it is? Why are certain proteins and probes fluorescent? What causes this light emitting property? We’ll have a look at these and more questions in this article. Start with a definition We’ll start with a…
Western blots can be ugly. I mean down-right, horrifically, wall-of-shame ugly. Not only can they be embarrassing to show to your colleagues, but the ugliness can obscure your results, making it impossible to interpret your data. Blotting consists of many experimental steps, which makes the technique naturally error-prone. Although standardized protocols exist, many fail to…
Sometimes a clever little trick for cloning comes along that makes you just give an appreciatory “ahhh.” For me, it was TA cloning. TA cloning is not a new technique (I am showing my advanced laboratory age here), but when I discovered it, the simple elegance of the technique made me pause and wonder about…
Digital PCR (dPCR) is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample. This method is similar to qPCR in the reaction assembly components and amplification reaction, but differs in the way the sample target is measured. Digital PCR is a…
Like most things in this world, fluorophores are mortal, and eventually your once bright fluorescent image will inevitably fade to black. This fading or ‘photobleaching’ of fluorescent signal can make imaging difficult, especially if you are trying to take quantitative images. Read below to learn what causes photobleaching of your fluorophores and how best to…
An ELISA (Enzyme-Linked ImmunoSorbant Assay) is a popular assay that uses antibodies and color change to detect proteins, peptides, antibodies or biomolecules in complex mixtures. ELISAs are popular because they are reliable, specific, easy to use, and can easily be scaled up to process multiple samples simultaneously. How an ELISA is Done: In an ELISA,…
We all know that stars are easier to see against the dark background of the night than they are to see against the bright sky of day. Well did you also know that the same may be true of your microscope specimen? Dark field microscopy is a popular microscope technique that makes your unstained transparent…
Every hard-core biologist knows designing the perfect construct can be a complex puzzle to solve. This challenge, if successful, can be extremely satisfying but can also drive you crazy for weeks. Luckily, Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute designed a new easy-to-use cloning method. Better yet, this system allows…
Although his name could fit in easily to the early 1980’s Hip-Hop Scene, Jerzy Nomarski (or ‘George’) was actually a Polish physicist with an interest in optical theory. Born in 1919, he eventually became a member of the Polish Resistance fighting in the Second World War. He was captured by enemy forces and held as…
While confocal microscopy seems to have become pervasive in cell biology, widefield microscopy techniques still have a special and important place. This month on the Microscopy and Imaging Channel, we’re focusing on widefield microscopy techniques: covering the basics of what these techniques are and when you should turn to them. What is this ‘Widefield’ you…
Bisulfite pyrosequencing is becoming a routine technique in molecular biology labs as a method to precisely measure DNA methylation levels right down to the single base. The technique allows for detailed and high resolution analysis of DNA methylation at specific genomic regions. How to detect the 5th base? Methylation of any of the four nucleotides…
You’re a senior grad student or postdoc, and you’ve done more PCRs than you can count. A new student has joined your lab, and you’ve been charged with training them on PCR. You don’t want to lead him/her astray, but it’s hard to remember the parts that you struggled with in your early days. This…
Phase contrast microscopy is a light microscopy technique which is primarily used to visualise live cells. Using various filters and condensers, the image produced by phase contrast allows us to see greater detail in live cells and can highlight aspects such as intracellular structures. Keep your cells alive! The best way to view cells is…
For those of us who work with Mus musculus or Homo sapiens, to name a couple of species, a few clicks on UCSC Genome Bioinformatics Site or Ensembl gets you the full and precise DNA sequence for any annotated gene in the genome. This luxury is not in place for all species however; many of…
Biologists have long appreciated the complexity of genome organization, but until recently lacked the tools to discern the intricacies of this puzzle. Now, thanks to some handy cross-linking, careful amplification, and (of course!) next generation sequencing, teams from Massachusetts are taking us down the rabbit hole, with some surprising findings from Wonderland. Bend Over Backwards…
Discover the magic of toluidine blue – a polychromatic dye that changes color depending on which tissue component it is staining.
Blunt-end cloning involves the ligation of DNA fragments – usually between a plasmid vector and an insert – whose terminal ends are not “sticky”. Performing these ligations is notoriously difficult, particularly with large DNA fragments. But it is possible. And in this article I’ll give you some tips that I hope will increase your chances…
In studies of RNA abundance and gene expression, no one technique can answer all of the questions that need to be asked. So it is necessary to use a variety of experimental methods in concert. Two RNA detection and measurement techniques that complement each other well for this purpose are RNA Fluorescence in situ hybridization…
Get introduced to some of the special stains for histology and learn some top tips for getting great results.
If you’re picking up a list of available plasmid vectors for the first time, it can be mind-blowing to decide on the best one for your experiment. What cloning sites do you need? What restriction enzyme sites? What insert size is possible? Let me help you with some pointers about what factors to look out…
It’s 11 PM…do you know where your samples are? If you dread the thought of having to hack through ice, snow and the bone-chilling depths of every freezer to locate them, flirt with frostbite only once. Better yet, never hunt for missing lab samples again! Here are some useful ways to keep track of every…
Looking for paraffin alternatives for tissue embedding? Find out the benefits of cryo and resin tissue embedding and how they work.
The lac operon is an amazing tool in molecular biology. It has been used for decades to turn on protein expression in an inducible manner with IPTG. The result is synthesis of vast amounts of protein to be used as you wish. While the lac operon is an amazing tool for protein production, it is…
Removing proteins from acqueous solutions can be a pain if you have loads of samples to do. This article explains how to prepare the DIY phase separating gel to easily and effectively separate two-phase mixtures, saving you time and money.
Single Nucleotide polymorphisms (SNPs), colloquially pronounced ‘snips’, are the most common type of genetic variation in people. By definition, a SNP represents a single nucleotide variation at a specific location in the genome that is found in more than 1% in the population. For example, a SNP can replace the nucleotide cytosine (C) with an…
Most of the microscopes you will encounter in your laboratories will be ‘upright’. In other words, they are assembled (from top to bottom) in the order of; eyepieces, objectives (on revolving nosepiece), stage, sub-stage condenser, diaphragm and base. However, there are two other types of light microscopes you will perhaps encounter (and use) and it…
Scientific creativity and repetitive tasks do not go well together. There is nothing worse than having to repeat the same task over and over again – I know somebody who quit science because of this and became a castle curator. So how do you minimise the amount of one of the most repetitive tasks –…
PCR has become the tool of choice for molecular diagnostics and is now a staple platform in any laboratory setting. The versatility of this method has led to a myriad of spin-off techniques, including probe-based quantitative PCR (qPCR). This method effectively combines PCR amplification and detection into a single step to measure the specific amount…
Counterstaining can have a big impact on your histology result. This short guide will introduce you to some available counterstains providing you with a few more choices.
Achieving a good immunohistochemistry signal-to-noise ratio involves many factors, including a good blocking protocol. Read on to learn about blocking non-specific staining in IHC.
Plasmid mapping and DNA annotation software is pretty abundant these days. A quick Google search brings up dozens of hits – but how do you know which one to use? If you are like most molecular biologists, you probably use the same software your colleagues do—usually it is either the stuff that gets passed down…
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