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So, you’ve spent time planning your high-throughput sequencing experiment. You’ve chosen how many replicates to use, deliberated about sequencing depth, and kept everything RNase-free. Now you have many gigabytes of data available. What’s next? While the first step of RNA-Seq analysis is aligning your sequencing reads to a reference genome, first you need to get…
Science outreach is a great way to energize yourself to work better at the bench. If you are curious about doing outreach and want to know how to get the ball rolling, I can suggest places to look for people and events to connect with. Like a knitted sweater, you only have to find one…
Welcome to the microbe series where we have a very exciting line-up planned over the coming months. Here we will talk about everything microbial, including the uses of microbes in industry and medicine, emerging pathogens, diagnostics, and much, much more! Let’s kick off this series with an introduction into these wonderful, yet sometimes nasty, organisms…
So, you have successfully cloned your gene of interest and are eager to purify buckets of protein. No matter your eventual application—kinetic experiments using a SPR instrument, structural analysis using X-ray crystallography, or any other experiment—you’ll need to express your protein first. Now, it’s time to put your expression plasmid into E. coli and get…
Surface plasmon resonance (SPR), a label-free, real-time way to examine protein binding and other molecular interactions, is getting easier as manufacturers have streamlined SPR instruments and supporting software. But problems can still arise. Troubleshooting Your SPR Assay Here are some common issues and suggestions to solve them: Inactive Targets Your target protein may have become…
If you have ever imaged biological samples, you have likely encountered autofluorescence. That pesky background coloration you see under the microscope, which can make it difficult to distinguish your actual signal from the noise.1 When you are trying to look for something as delicate as RNA, you don’t want to be hunting for your signal…
To many users, the flow cytometer is a magic box: put in cells, get out data. You click the button to tell it which colors to look at without much thought about how the machine does this. However, not all fluorophores are created equal—some configurations might exclude the spectrum you’re really looking for. Here’s a…
In austerity times, nothing is in excess. Apart from saving reagents, which can be refilled with extra financial injections, there is a commodity that cannot be easily resupplied – tissue samples! If, like me, you have experienced the fear of not having enough sample for performing a qPCR, western blot, and conventional PCR from the…
You already learned the basics of column packing. When moving to more automated system using low pressure liquid chromatography systems, you can use pre-packed columns. But in order to compare several resins in specific conditions, and also to save money, you might need to pack your own low pressure columns. The art of packing a column…
P-value abuse directly contributes to one of the biggest problems facing the scientific community: the prominence of false-positive results in the published literature. Contrary to popular interpretation, the p-value doesn’t indicate the likelihood that the observed result was due to chance. There are important qualifications to p-value interpretation. Moreover, the p-value cannot directly speak to…
While using human clinical samples in your research can provide robust and heterogeneous results applicable to larger portions of the population, working with these samples presents its own set of challenges. Here are some tricks I have learned to help isolate and grow your cells of interest while eliminating stromal, blood, or other undesired contaminants….
Try to recall the last time you were not alone in the lab at 11pm on a Friday night running an experiment. If you found that to be difficult, then this article is definitely for you. Here are ways to avoid feeling isolated during your Ph.D. studies. Build a Network of Friends and Colleagues Building a…
So, the genome-wide association study (GWAS) data for your disease of interest was published, and it has thrown up some very interesting associations. However, at this stage, bear in mind that this is only an association. Your project is to provide the link between the GWAS single nucleotide polymorphisms (SNP) and pathological changes. Where do…
What is Adeno-Associated Virus? Adeno-associated virus (AAV) is a popular gene therapy vector. Different AAV serotypes infect different cell and tissue types with varying efficiency, so it’s a good idea to select the serotype most relevant to your work. The most commonly used AAV serotypes are 1, 2, 5, 6, 8, 9 and DJ, although…
Anytime lab processes get automated by a sophisticated scientific instrument, there can be a “black box” effect, leading users to wonder what’s going on in there. For DNA electrophoresis, it’s no different. It’s easy to see what’s happening in a manual gel, but the automated gel-based DNA size selection platforms can be more mysterious. Automated…
Are you preparing to set up live cell imaging experiments? You’ve got all your cell lines, antibodies, reagents, and protocol ready. You just want to wake up in the morning and enter into that dark room. Well, think again!! As we (I mean the cell biologists) always say, happy cells mean happy life. You have…
Running a pulsed field gel can be exciting. It isn’t often that you get to visualize such large pieces of DNA. However, it can also be extremely frustrating. It isn’t uncommon to wait 24 hours only to find out that your DNA was degraded before you started the run. Then, you have to start all…
Anchorage-independent assays test the ability of cells to grow independent of a solid surface. The assay is used to check the malignant potential of cancer cells. Cancer researchers generally do this experiment for any kind of confirmation of the oncogenic potential of an oncogene or a tumor suppressor in cancer cells. However, we do encounter…
Let’s face it, when you leave the lab for the day, your mind is still racing with ideas and questions about your experiment: how to fix this or what went wrong or what does this data point even mean. It can be difficult to actually shut down and decompress when you walk out of the…
The elusive manuscript. It’s what we, as scientists, build our kingdoms on—throwing ourselves into our research, hoping to feel our time in the sun when it all comes to fruition in the form of that glorious body of work. But…what how do you determine who should share in that sunshine? Should you always put your…
Surface Plasmon Resonance (SPR) is the gold standard for measuring biomolecular binding without the need for labeling (i.e., label free detection of kinetics). SPR is especially valuable because it doesn’t just provide information at the start and end of a binding event, but can be used to follow association and dissociation kinetics of biomolecules in real-time….
Presenting science concisely poses many challenges: How do you say enough without saying too much? Are you capturing the main points? Does this research paper abstract attract the reader’s attention and make them want to read your paper? That last question is the most important and the most overlooked one. And to address it, many…
When I was being trained in microbiology as an undergrad, one of the first skills I acquired was the ability to quickly compare and visualize amino acid sequences using BLAST and ClustalW. 15 years later, those two programs have done nothing but improve by expanding the data contained in these databases and simplifying the user…
If you’ve been keeping up with our recent series of articles, welcome back! If not, you can catch up on how fluorescence works or what not to do with your flow experiment. In short, we have been discussing fluorescent labels and their role in flow cytometry. Today, I’ll round out our discussion by touching on…
Whatever molecular biology techniques you use, at some point you will have to clean up your DNA samples to remove things like buffers, contaminants and nucleotides from you precious sample, so that you have perfectly pure DNA for your downstream experiments. Magnetic beads are one DNA cleanup option. They are simple and effective—and their…
Image analysis can be biased. Discover the three main steps to image quantification and learn how to quantify images in an unbiased way.
This article provides interesting insider PubMed tips, including how to use search filters and how to set up automatic search alerts.
The only constant with microscopy imaging is variability in both color and image quality. You only need to look at images in journal articles, posters, around your laboratory, or compare your images with a colleague’s—the evidence is staggering. Interestingly, variability doesn’t generally come from the digital camera, rather it comes from our use of imaging…
In the midst of all the cool new sequencing techniques and technologies out there today, you may have overlooked the tried and true method of Shotgun Sequencing. What is Shotgun Sequencing Anyway? Shotgun sequencing gets its name from the concept that a large sequence is essentially broken up in to many, many smaller pieces, similar…
In my last article, I discussed how to best keep your lab’s HPLC running smoothly. However, even the best-maintained HPLCs and columns need periodic cleaning. Today, I’ll describe how to identify and troubleshoot a clogged HPLC column. Columns ARE Finite First of all, it’s important to realize that columns do have a finite lifetime. The…
Flow cytometry. Some people love it—most hate it—but all can agree that it is one of the most powerful analytical tools immunologists possess. Here’s a quick refresher: as the name suggests, flow cytometry measures the physical and chemical characteristics of cells. This is accomplished by fluorescently labeling cell surface markers/proteins using antibodies conjugated to fluorophores….
You can create stably transformed plants expressing your gene of interest; be it for the subcellular localization of your protein or simply for the in planta protein expression and purification. Whatever it is, you can do wonders with plant transformation. Sound difficult? It isn’t. Just like there are millions of microbes that interact with us,…
Communicating your science to a lay audience is different than giving a talk to other scientists. An urban legend says that when Michael Faraday verified the relationship between electricity and magnetism, he was asked to present his evidence to the prime minister of England. So, he had his coils arranged and he just moved a…
If you work in the field of molecular biology, there is hardly a day that goes by that you don’t use nucleotides. But beyond the use of the four well-known deoxynucleotides in PCR, you can use nucleotides for several other applications. For example, kinases and phosphatases use nucleotides as substrates, and phosphotransferases transfer phosphate group…
Like all technical fields, molecular biology contains a very robust “theoretical” realm and an equally robust “practical” realm. Unfortunately, these two existences don’t seem to overlap as often as we’d like. Consider, for example, a simple Western blot. While an antibody interacting with its target on a membrane seems pretty straightforward, there are numerous other…
At the heart of cloning are restriction enzymes. Restriction enzymes are a common tool in any molecular biology lab. Need to know how large your plasmid is? Cut it with a restriction enzyme. Need to chop your genomic DNA into smaller pieces for a southern hybridization or to prepare a library? Use a restriction enzyme….
Have you ever entertained the idea of learning to program? Have you tried but felt discouraged by the overwhelming amount of information out there? If you answered yes to both of those questions, I encourage you to try again with the following resources. Computer science is one of the best subjects to self learn. …
DNA shuffling uses PCR technology in a very creative way. It allows you modify your protein to make a new protein you want. You can evolve proteins in microcentrifuge tubes on your very own lab bench. Isn’t that fantastic? DNA shuffling is also a very powerful technique for directed molecular evolution. W. Stemmer first used…
You have probably run a standard agarose gel hundreds of times. They are great for visualizing small DNA fragments up to 10 kb, but what if you want to examine really large pieces of DNA or even whole chromosomes? This is where pulsed field gel electrophoresis (PFGE) comes in! While the equipment required to run…
As science is becoming more interdisciplinary, the tools we use to answer questions are also crossing party lines. Case in point: flow cytometry. Once a tool only used by “real” immunologists, flow cytometry is fast becoming a method by which numerous questions can be answered, from the length of a cell’s telomeres, to the state…
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