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Microarrays are one of the most in-depth ways of determining cellular gene expression levels of thousands of genes simultaneously. They are able to help determine: Getting good microarray results starts way before analyzing your data, however. A crucial first step in setting up a good microarray experiment is extracting high-quality RNA from your cells. In…
For applications such as site-directed mutagenesis, it is often recommended that you use a proofreading polymerase (also known as high-fidelity polymerases) to minimize the risk of introducing unintended point mutations. But what is a proofreading polymerase? What makes them different from other polymerases? And when should you use them? Read on to learn more… What…
Most site-directed mutagenesis protocols strongly recommend that you use only PAGE- or HPLC-purified primers to mutate plasmid templates. Using purified primers is supposed to minimize the introduction of unintended mutations, thus drastically improving the probability of generating your desired mutant. However, specially purified primers can be extremely expensive, and take longer to synthesize than standard…
RNA interference (RNAi) may have originated as a defense mechanism to protect cells against foreign genes introduced by viruses. This concept has since been put to use to create a powerful experimental tool for investigating gene function in organisms. Small-interfering RNA (siRNA) libraries for investigating genome-wide function can be produced by chemical synthesis of probes…
ELISAs (enzyme-linked immunosorbent assays) are often used for detecting and quantifying substances such as peptides, proteins, antibodies and hormones in research and diagnostics. Today, a wide range of ELISA formats exist to suit your needs e.g. indirect ELISA, direct ELISA, competitive ELISA and sandwich ELISA. While it has become easier to perform ELISAs, thanks to…
Although the microscope is probably the most commonly used biological instrument, it is frequently used improperly. The rate-limiting step to getting high quality microscopic images is illumination of your specimen. When you examine a specimen under the microscope, the intensity and distribution of light must be clear and equal to enable you to evaluate all…
The presence of supercoiled plasmid DNA on a gel can be inconvenient for molecular biologists, especially beginners because it is easy to misinterpret. Read our top tips how to recognize supercoiled DNA and keep it from derailing your cloning experiments.
If you’re starting your PhD or post-doctoral work, chances are you’ll need to use a light microscope at some stage during your research. Some of you may be seasoned microscopists. For many of you though, this might be the first time you’ve ever plugged in a microscope, or at least the first time you’ve used…
ChIP-seq is a wonderful technique that allows us to interrogate the physical binding interactions between protein and DNA using next-generation sequencing. In this article, I’ll give a brief review of ChIP and introduce the chromatin immunoprecipitation sequencing technique (ChIP-seq), which combines ChIP with next-generation sequencing. What is chromatin immunoprecipitation? Chromatin immunoprecipitation (ChIP) allows us to…
If your immunohistochemistry is not working quite as expected, don’t fret. Check out this comprehensive guide to troubleshooting immunohistochemistry.
Have you ever wished you could transfer the same SDS-PAGE gel twice? Sometimes, when you are blotting for many different proteins of similar size, stripping and reprobing multiple times can become impractical. Here’s a simple diffusion transfer method that can be used to generate duplicate membranes from a single gel: Protein transfer by this diffusion…
Phosphorylation is one of the major post-translational modifications that regulate the activity of a protein. Around a third of human proteins are believed to be phosphorylated, and so the kinases and phosphatases that mediate protein phosphorylation are of major interest to biomedical researchers. However detecting protein phosphorylation can be difficult, particularly from cell extracts. Phospho-specific…
Back in August I shared my training regimen for guesstimating the OD600 readings of microbial cultures with superhuman accuracy. Although my method is effective, I will admit that it has two shortcomings: you need to make a separate standard curve for each container type, and guesstimation is not an officially sanctioned scientific method. But now,…
If you use a human cell line in your research, have you wondered where, or who, it came from? I never gave it much thought, until I read Rebecca Skloot’s book, The Immortal Life of Henrietta Lacks. In 1951, cervical tumour cells were taken from Henrietta Lacks and put into culture, to divide endlessly and…
For a long time we’ve been able to pinpoint the subcellular location of proteins, and the advent of FISH (Fluorescence in situ Hybridization) allowed us to locate the position of genes in the nucleus, but recent advances in RNA FISH are making it easier and easier to collect the same data about individual messenger RNAs….
Previously, I introduced the DNA microarray technology and described the principle behind it: hybridization between the nucleic acid sequence from the biological samples being examined and a synthetic probe immobilized and spatially arrayed on a solid surface, the microarray. In this article, I will explain how these probes are designed and positioned on the array. I…
Do you want to immunoprecipitate (IP) a protein with a molecular weight that is anywhere near 55 kDa or 25 kDa? Then you have an irritating problem to deal with: antibody co-elution. But don’t panic, we have six strategies for dealing with your new problem. The Problem: Typically, the IP antibody is bound to Protein…
Working with RNA is definitely an acquired skill. It’s a lot more finicky than working with DNA, and requires careful attention to detail in order to avoid disastrous through RNase contamination. Here are a few common ways to lose your hard-earned RNA: 1. Don’t keep everything on ice Keeping the temperature of all of your reagents cool is…
Restriction enzymes are a basic tool in the molecular biologist’s arsenal. They’re super easy to use, and virtually essential for cloning and other applications. Restriction enzymes are also a great example of a perfect “tool” from nature that scientists have co-opted for their own use. Here are a few fun facts about restriction enzymes that…
I once knew a postdoc who was absurdly accurate at guessing the optical density of microbial cultures. I was jealous – imagine how much time I would save if I could spec my cultures just by looking at them! Since I lacked the innate optical talents required to compete with my friend, I developed a…
Transforming yeast with DNA is a very similar process to transforming E. coli, but with just enough differences to trip you up if you let your attention slip. Whether you’re doing a yeast two-hybrid screen, or using yeast as a model system, here are a some mistakes to to avoid… 1. Forgetting to add single…
Site-directed mutagenesis studies can be extremely useful for elucidating the function of a gene or protein, or for creating variants of an enzyme with new and improved functions. There are now many approaches available for generating site-directed mutants, whatever your purpose. In this post I’ll summarize three techniques that will enable you to produce a…
One of my colleagues, a very good molecular biologist, told me that the only time she uses chemistry is when she needs to calculate molarities. I, of course, scoffed at this statement, and tried to remind her of all the chemistry she uses daily. True, I may be a bit biased since I am a…
Here’s an all-too-often repeated scene in the lab: First thing in the morning, you approach the 37°C incubator with trepidation, open it and through one half-open eye you take a look at the LB plate that you spread your ligation-reaction-transformed E.coli aliquot onto. Looks good – thousands of colonies. Emboldened, you take your “no ligation…
Sepharose beads are porous, which gives them a high surface area for interaction with proteins and allows them to hold a lot of liquid. This is perfect for the application that they were originally designed for: purifying milligrams of protein in columns. When immunoprecipitation (IP) – a small-scale technique for pulling specific proteins out of solution using…
Virtually every research scientist has a use for sterile technique in the lab, whether you study infectious microorganisms, do tissue culture, or use E. coli for cloning. Good sterile technique is a basic lab skill required to avoid contamination of your materials and experiments; and fortunately, the principles are simple to learn and easy to…
At the end of my last article, I provided some practical tips and tricks for working with enzymes at the bench. Now, we’ll cover one of the cornerstone techniques of enzymology work: the enzyme assay. Starting with the simple assays and eventually working our way to the more complex, this article introduces the principles of…
A partial digest – typically done when you only want to cut one of two or more restriction sites in a DNA – can be a frustrating procedure to execute. The best advice anybody can give about partial digests is to avoid having to do them. However, there are times when there just aren’t many…
Western blotting is a common lab technique used to detect and analyze proteins. It also happens to be a really long and complicated procedure, with many steps along the way that are easy to mess up. How do you make sure that your Western blot is successful? Avoid the following five ways to destroy your…
In Part 1 of this series, we began our journey into the fascinating world of enzymology. We looked at the most basic concepts of what an enzyme is and the incredible jobs it can do. In Part 2 of “Working with Enzymes,” I will look at some things that you should keep in mind to…
In parts one and two of this series I described how semi-permeable membranes and precipitation methods could be used to concentrate your protein-of-interest, but there is one more method that you may not have thought of for protein concentration – chromatography. While chromatography resins are an obvious choice for protein purification, they can also be…
While precipitation is an obvious choice for concentrating DNA and RNA samples, it can also be an effective way to concentrate proteins. Here in installment two of this three part series, I describe the two most common methods for protein precipitation – ammonium sulfate and trichloroacetic acid. Background Precipitation of proteins occurs primarily by hydrophobic…
This is the first of a three part series describing some of the most common methods for concentrating proteins. In later installments I’ll discuss using protein precipitation and chromatography to concentrate a protein. However, here I’ll detail the most popular approach – semi-permeable membranes, used for both dialysis and commercial protein concentrators. Structure of the…
Protein kinases and protein phosphatases phosphorylate and dephosphorylate a plethora of proteins. They are responsible for regulating the majority of cellular activities. Because of their importance, they can seem intimidating to tackle as a research project. At the end of the day however, kinases and phosphatases are- simply put- enzymes. Therefore, you can standard enzyme…
If you purify proteins expressed in E. coli, then you’re probably familiar with this scenario: you come in bright and early in the morning and inoculate your large flasks of media with the overnight culture, start shaking them at 37 °C, and now you wait. And watch. And wait some more. You can’t venture far,…
I learned most of my molecular biology skills in the first lab I worked in almost 10 years ago. I realized recently that I was in desperate need of a refresher course, so I did a little bit of reading to see if I could improve the efficiency of my cloning reactions. In the process,…
Chris Dieni wraps up his series on the Basics of Protein Phosphorylation with this final part on further reading and resources available.
Chris Dieni explores the promiscuous yet intriguing nature of protein phosphatases
CIP has been around for a while, but there is a better alternative.
Using UNG is a great trick for PCR amplicon decontamination, but is it any good for rtPCR?
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