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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…
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…
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…
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…
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….
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…
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.
In the first part of this article (you can read it here), we looked at clipping and saturation in terms of microscope images, followed by a definition of Dynamic Range and an introduction to Bit Depth. Intrascene Dynamic Range The dynamic range which can be detected at the same time in the same field of…
Radioactive protein labeling is not as common as it used to be. With the advent of modern protein labeling techniques, such as fluorescence, radioactive labeling has largely fallen out of favor. However, radioactive protein labeling is still a very useful technique and is often superior to more modern labeling techniques. Radiolabeling can provide a…
With the current proliferation of new dyes and instruments that can detect many colors simultaneously, it seems like an entire rainbow is at your disposal for your flow cytometry experiments. And we know that when designing a polychromatic flow cytometry panel, more is often better – right? The more antigens you can detect, the more…
We are pleased to announce that the famous maid of microbiology, dear old Aunt Yersinia, has agreed to start writing a microbiology and molecular biology advice column for Bitesize Bio. She will be free to answer your most pressing questions sent to: auntyersinia@bitesizebio.com By way of introduction, Aunt Yersinia is bestowing 8 spores of knowledge garnered…
Perfect your subcellular fractionation in the lab by understanding how these protocols work. Plus, discover the various kits for organelle separation.
Around and around the cell cycle goes, where it stops, nobody knows. Unless you have the right tools to analyze DNA content, that is. The DNA markers propidium iodide, Hoechst and DAPI are commonly used in flow cytometry to analyse a cell’s DNA content. Although they are simple to use, they do have disadvantages. Figure…
One of the most exciting aspects of being a biologist is getting opportunities to examine how and why living organisms behave the way they do. We have technology that enables us to obtain images at sub-cellular levels, and the skills to work directly with the micro-environments essential for the progression of life. However, at the…
A wide variety of enzymes are available for PCR and RT-PCR and the optimal choice depends on a range of factors specific to your experiment. Some of these factors will now be explored to help you to make the most suitable and cost effective choices when ordering. PCR Type and Other Factors to Consider First,…
Ever tried to turn the volume all the way up on a small radio or small stereo system? (Hopefully you have not tried it with earphones in!) Notice how, after some point, the sound didn’t get any louder- it just got more distorted? That’s because you’ve hit the ceiling of your machine’s dynamic range. It’s…
What if there was a way to take the power and speed of a flow cytometer and couple it with the resolution of a microscope? Imaging flow cytometry does just that! Flow cytometry is a very powerful tool for the complex characterization of cells and cell populations but flow cytometers can also be thought of…
If you are struggling to optimise your Western blot protocol, one step to consider is the equilibration of your gel and membrane before transfer. Wondering what this step achieves and whether it’s necessary? You’re not alone! I did dozens of Westerns without ever bothering to equilibrate before I realised that it was having a big…
You are sitting in a seminar when you glimpse it: the figure that beats all other figures – a beautiful contour plot – and you realise that a similar figure is what you need to publish your paper in a high-ranking journal. You know the basics of flow cytometry, but admit you need a little…
So you’re designing a new experiment that requires PCR quantification. You used to have only one method to choose from, but now you have two – Quantitative Real-Time PCR (qPCR) and Digital PCR (dPCR). Which one is right for your application? Both methods have good quantification, sensitivity and specificity for most applications. They are compatible…
Stimulation of cells/tissue with a given stimulus (e.g., a cytokine) is a common experimental setup in any cell biology lab. The cellular response to the external stimulus e.g., the activation/deactivation of intracellular signaling pathways and/or the secretion of proteins is often the research goal, and there are a number of different methods that you can use to analyze such…
Bitesize Bio has had a lot to say about RNA isolation, mainly because it is one of the most anxiety-producing requirements for molecular biology; especially when you are first starting out (although isolating proteins from complex samples like soil and stool is far more difficult, let me tell you. But that’s a future post.) We’ve…
If you want to visualize elastic fibers in your sample, you need to use Verhoeff-van Gieson stain. Find out more about this stain, including how to use it.
You spent the last few weeks tweaking your Co-immunoprecipitation conditions, testing different antibody/bead combinations, and sampling a panaply of solutions and FINALLY! You have your Co-immunoprecipitation (Co-IP) elution… Now what? Well, you have a few choices. It really all depends on what you need know about the proteins in your elution. Do you need to identify…
The flow cytometer that we have all grown to know and love may have only come into its own in the 1990’s, but who would have known that the first cell sorter was invented as early as the 1950’s? With the recent death of one of the key developers of fluorescence activated cell sorting (FACS),…
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…
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