...with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements, the cytometer...precisely measure your cells of interest. In many experiments you’ll be working with a heterogeneous cell population...describe the shape of a histogram, which graphically represents the time and intensity of the cell’s illumination...cytometry can be used for virtually all kinds of experiments and research fields, for any experiment, your...cells, the single cell gate to exclude duplicate events, and the Live/Dead gate to focus your analysis ... As an example, we’ll use one of my previous experiments where I harvested a melanoma tumor from a C57BL...my lymphocyte population (10.7% of all recorded events) based on their expected size. The strong signals...

...work with your colleagues, supervisors, clients or even students, your ELN should allow you to collaborate...paper-based record keeping barely copes with the growing amounts of digital data generated in labs. While many labs...different ELN features, ranging from whether respondents saw them as not important to very important. ...explored the barriers to adoption within academic environments to better understand the issues labs are facing...limited budgets, “a large percentage of survey respondents indicated that cost was a significant barrier...system within their institution. In the study, respondents were also asked about how important ease of use...from paper to digital for example): “… 99% of respondents indicated that ease of use would influence their...

... your protocols, designed your experiments, and prepared your reagents. You’re going to engineer the best...assemble up to 6 different fragments in a prescribed order. These fragments can be pooled in one tube ...PCR to generate DNA fragments with 15-40 bp of homology at their ends. These fragments are then combined...methods are more amenable to combining multiple DNA fragments than others. In today’s post, we will focus on...in an hour or less. The ligations combine the fragments in an order determined by their homology. You ...cloning process. Time spent actually doing the experiments are also generally comparable so the best way...optimize reagent concentration, or purify DNA fragments from gels. If you’re relatively new to the cloning...

...problem.  Pre-block with Fab fragments (Figure 2C) Speaking of Fab fragments… Another strategy for species-on-species...been bound up by the Fab fragments. Note that you should plan to use Fab fragments that are from the same...should not recognize Fab fragments from the same species and 2) these fragments will be more likely to ...include no primary antibody controls in your experiments. If you do find high levels of background staining...that is directly conjugated to your reporter circumvents the issue. However, directly conjugated primary...pre-treating the tissue with unconjugated Fab fragments that recognize the endogenous IgGs (Lu and Partridge...Partridge, 1998). After washing away unbound fragments, you can then apply your primary and secondary antibodies...

...Complex experiments Finally, additional controls may be required for more complex experiments. A multiplexed...helpful to focus on something controllable, like experiments (and their controls!). This blog post will discuss... the ins and outs of controls for biological experiments, starting with general controls and then moving...design thoughtfully and thoroughly controlled experiments.  Broadly useful controls Positive and negative...negative controls Most biological experiments require positive and negative controls to ensure proper interpretation...group.     Figure 1: A, Most biological experiments will benefit from positive (+) and negative (...group and C, technical replicates or repeated measurements will increase the power of the results. In this...

... antibodies in plants using syringe agroinfiltration." Recombinant proteins from plants. Humana Press,...candidate at Northeastern University. Working with plants doesn’t always have to be a time-consuming process...reproducible. Prior to creating transgenic cultures or plants, our transient expression method enables us to ...leaf transformations The development of transgenic plants is a common problem in non-model organisms. Therefore...choice for investigating gene function, as these experiments can be done in days to weeks instead of months...roseus. A common method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium infiltration...more easily quantify the effect of the different treatments on transformation success. Some reporter gene...

...curate reagents from these emerging organisms so scientists can easily find the useful new reagents that...Cnidarian-Dinoflagellate symbionts (Virginia Weis) Sea anemone Aiptasia - algae Breviolum minutum symbionts are important...Westwood) Dodders are parasitic plants that steal all their food from other plants and are unusual enough to...between gene function and phenotypes in diverse plants, animals, microbes, viruses, and fungi for which...for scientists and educational programs for K-12 students. You can see why I was excited to be invited to...at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers were...and Asclepias curassavica (Gerog Jander) Milkweed plants are known to many as the only Monarch butterfly...

...many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm...misfolded FP-fusions on quantitative imaging experiments and cell physiology in general. Pitfalls of Fluorescent...apparent and can confound quantitative imaging experiments or even negatively impact cells (see Figure 1...sequences, which often have absolute position requirements. For example, a KDEL ER retrieval sequence only...that FPs are often poorly suited for cellular compartments other than the cytoplasm. Generally, FPs evolved...in or were engineered for use in cytoplasmic environments. However, approximately 40% of human (and most... localize to chemically distinct subcellular environments, including the organelles that compose the secretory...

...conditions. Purifying DNA fragments Once you’ve generated your DNA fragments, it is always a good habit...digested fragments or PCR products from a gel. You will avoid contamination from other DNA fragments and you...nanodrop to assemble the  fragments based on their molar ratio. Assembly of the fragments There are multiple ... way to do it (i.e. avoid assembling too many fragments at once, if it is possible). There are many softwares...could affect or delay your experimental work. Fragments generation After you’ve designed your cloning ...cloning scheme, you’re now ready to generate fragments. You can generate the parts of DNA that you want to assemble...between the different enzymes. Always check the fragments size of your digestion on an agarose gel. If the...

...neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed below ... you are measuring the events-of-interest. Sensors suitable for ER measurements include: CEPIA (additional...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...slower). GCaMPf may be suitable for very rapid measurements, like action potentials in neurons. jGCaMP7 ...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ... the (1) binding of calcium, (2) allosteric rearrangements of the sensor, and (3) subsequent change in...you combining your calcium reading with other measurements, specifically fluorescent ones? Many of the ...

...uniquely challenging to apply in dicotyledonous plants, or dicots. But Jae-Yean Kim’s lab recently "broke... strategy uses new combinations of PE protein components (Figure 1), an altered epegRNA delivered with...chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene to confer resistance..., two weeks after herbicide was applied to both plants. Adapted from Vu et al., 2024 bioRxiv, under CC-BY-NC-ND...both tomatoes and Arabidopsis, suggesting the improvements may be broadly applicable to dicot species. .... In addition, they generated offspring tomato plants that inherited the genomic edit but not the editing...desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https://doi.org/10.1038...

...electrophoresis. Smaller fragments migrate quicker than larger fragments. These fragments are quantified over...the DNA such that smaller fragments are quantified first before larger fragments. Figure 1: The quantification...units over time. Smaller fragments migrate more quickly than larger fragments. Image from OpenWetWare....your DNA prep and whether there are any other contaminants. Nucleic acids (DNA and RNA) absorb maximally...quick and simple and doesn’t require any special reagents. However, there’s limited sensitivity at low concentrations...piece of DNA. This method is best used for DNA fragments (like a PCR product). This method also gives an...a few minutes per sample. During the run, DNA fragments move through a micro- or nanofluidic channel and...

...literature. Data reported from flow cytometry experiments can be a little challenging to understand if ... and output is generated through measurement of events. An event is something that passes through the ...axis to generate a dot plot (Figure 1). Clustered events are called a population.                           ...left, it might make more sense once you realize events are plotted on an invisible Cartesian plane (right...real-world data is far messier than biorender plots, quadrants can still help me as a reader if I'm struggling...strategy from Barlow-Anacker et al., 2017, with quadrants to help visualize the different populations. Used...populations; this indicates the percentage of total events contained in the gated population. Heat maps are...

... protocol or reagents, do you understand all of the steps? Do you know how the reagents have been validated...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...couple of months and requires that all of the experiments have been completed. Therefore, to be ready to...postdoctoral mentor. A good mentor typically writes the grants that fund your research, guides the research directions...provide expertise in designing and interpreting experiments, as well as career guidance. In my own experience...the methodology and can have confidence in the reagents and outcomes. This is preferable to finding out...work for your specific assay. "Quick and dirty" experiments often eat far more of your time as they can result...

...fluorescence imaging strategies use widely varying amounts of excitation power (Shaner et al, 2008). In confocal...to predict how fluorophores perform in ‘real experiments’, it is advisable to perform time-lapse imaging...with low excitation power. By repeating these measurements for different fluorescent proteins and by comparing...protein distribution. Therefore, photostability measurements can be performed with soluble fluorescent proteins.... It should be noted that the photostability measurements reported in the literature are performed in ... the experimental designs used to make these measurements and hope this information can help guide your...evolves over time. The second issue is that, in experiments that measure photostability, high excitation ...

...overhangs can be used to assemble multiple fragments - up to 10 fragments are commonly assembled in a single ...increased number of fragments, or the ligation of very small/very large fragments, these problems can ...homology at the ends of DNA fragments to specify assembly order, so fragments with 5’ or 3’ sequence homology...they can be customized to direct assembly of DNA fragments. When designed correctly, the recognition sites... These overhangs specify the desired order of fragments, and the loss of enzyme recognition sites after...BpiI/BbsI to efficiently assemble up to six DNA fragments at a time. As with all Golden Gate-based methods...outside their recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled...

...site-specific recombination events. The system consists of two components derived from the P1 bacteriophage...present; intramolecular events happen with greater frequency than intermolecular events, but any two sites ...series, we examined a number of important plasmid elements – promoters, origins of replication, protein tags...recognition site. The P1 bacteriophage uses these components as part of its natural viral life-cycle, and ...and researchers have adapted the components for use in genome manipulation. Cre recombinase, originally...allowing for broad applications in a variety of experiments. LoxP (locus of X(cross)-over in P1) sites are...modification can be restricted based on experimental constraints. This has been used for a wide range of purposes...

...money on optimizing experiments, to false conclusions.  Figure 1. Potential points where DNA errors...shifts, missing elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences...an important part of maintaining the quality of reagents, especially shared ones.  The importance of QC... propagation of plasmids in bacteria — has many points at which small errors can be introduced. Eventually...introduced, indicated by exclamation marks. Error points include places where DNA is being created, such...tags, promoters, and other critical functional components. Fun fact! Some older plasmids in our collection...each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated...

...link together multiple fragments, allowing ordered assembly of multiple components into a destination vector...suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...(here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly into a Cas9...and thus the highest frequency of genome editing events. Yamamoto Lab Multiplex CRISPR/Cas9 Assembly Kit... with the Gersbach lab plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase...clone your own entry vectors containing other Cas9 variants to use with the MuLE system. Multiplexing from...are also available from the Yang lab for use in plants. The mammalian multiplex systems use the Csy4 RNA...

...can vary "Lab assistants" usually wash glassware, prep media, etc. "Research assistants" are more involved... researchers, as well as graduate students (your teaching assistants), about their experiences. Research...investigator (PI) Runs the lab Writes a lot of grants Varying degrees of engagement with what happens...degree "Rotation student" - some PhD programs have students rotate through a few labs, ~10 weeks per lab, ...way to answer those questions. I’ve met graduate students who enjoy the thrill of acquiring a piece of knowledge...with your labmates and mentors Many of the grad students and postdocs I’ve met have done things that I ...about careers in science are geared towards grad students and postdocs—people who are a little ahead along...