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...including monitoring multiple cellular events such as the dynamics of subcellular structures and gene expression...other cellular proteins. BRET-based biosensors utilizing NanoLuc® luciferase Many intracellular sensors...luminescence. Intracellular Calcium Sensor Yang, J. et al. (7) wished to trigger intracellular Ca++ release...photoactivatable, optogenetic accuators to depolarize or hyperpolarize membranes. Traditional FRET reporters... sensor, membrane depolarization increases the BRET signal whereas hyperpolarization decreases the signal...orange fluorescent protein, CyOFP1. CyOFP1 was molecularly evolved from mNeptune2 through 33 mutations and...brightness and improved maturation time compared to similar orange fluorescent proteins. In their work, the...

... less selective if used in intracellular labeling because the intracellular compartment may contain cross-reacting...for the same clone. This can be particularly useful when designing larger multicolour panels, where different...approaches available broadly map onto the five pillars of antibody validation (see: Uhlen et al., 2016...for other sample types and for non-surface (intracellular) antigens. Unfortunately, antibodies frequently...protocol. This requires use of one of the five pillars of antibody validation, and it is recommended to...likely to express the target at a robust level, similar to that in samples of interest. If you are working...This can involve cell surface labeling and/or intracellular labeling (or both). It is important to test ...

...challenging to track intracellularly, but aptamers permit the study of intracellular RNA dynamics. Aptamers...used to investigate intracellular viral DNA dynamics in neuronal cells (Nilaratanakul et al., 2017) and ...be used to simultaneously target cellular RNA to specific intracellular locations using Cas9 and track ... satisfying ingredients reagents permeate the molecular biologist’s kitchen lab - and scientists using...molecule. Fluorescent RNA aptamers are used to track cellular processes by transforming cells with plasmids ...binding affinity to a specific target, and the molecular processes that make aptamer biology a powerful...a miniaturized version of Spinach that retains similar levels of fluorescence when bound to DFHBI despite...

...more efficiently amplified than larger constructs, but plasmids as large as ~6 kb can be amplified fairly...unmodified plasmid into a larger band (not shown). As a footnote; a similar approach can be used to identify... plasmid (or other). This is particularly relevant in cases where a large plasmid with multiple functional... to introduce a point mutation in a large construct. Particularly, if a pair of unique restriction site...the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data (see Xia...homologous ends recombine in vivo to give the final circular plasmid product pictured here. B) Screening of...portion of the plasmid containing the mutation). Similar results can be obtained with PCR screening using...

...combining them with a molecular species with high affinity for an endogenous molecular target, meaning the.... You may find yourself defaulting to the most popular or familiar options without even realizing it. ...attached to the target through binding of the molecular species. Administration of these dyes results ...fluorophores to enable highly specific visualization of cellular components, typically a protein of interest. Fluorescent... dyes/ligands Effects on protein function and cellular components: It's important to assess how dye-conjugation...affects protein function and its propensity to bind cellular components. Some dyes target specific organelles.... Other dyes may prove challenging to stain intracellular compartments in in vivo model systems, so extra...

...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University. No matter...for Biological Imaging and a Lecturer on Molecular and Cellular Biology at Harvard University.     Additional...Primer on the Molecular Expressions Website Other major microscopy manufacturers have similar information...color contrast, to an image and thereby provide molecular or structural specificity to the viewer. Finally...thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner. This is accomplished by...entire axial volume of a sample. Therefore, when a large number of optical slices are acquired all fluorophores...thin sheet of excitation light that propagates perpendicular to an imaging objective that collects fluorescence...

...practice in molecular biology and can be used in a multitude of scientific techniques such as molecular cloning... region of interest  Molecular Cloning Site-directed mutagenesis Molecular Diagnostics qPCR ...Protocol: How to Design a primer  Molecular Biology Reference Molecular Cloning Techniques ... chain reaction? If you have ever worked in a molecular biology laboratory you have likely done a polymerase...cloning or molecular diagnostics.       Steps of PCR The beauty of PCR is that it can amplify DNA using...quantity. End point PCR is most commonly used in molecular cloning, sequencing, and genotyping. It’s extremely...simply PCR, is used for an array of different molecular biology techniques and experiments. End point ...

...whole tissue (15).  CLARITY is also compatible with LSFM, a method termed COLM (clarity-optimized light-sheet...seen some major advances in light microscopy, particularly related to the neurosciences.  These developments...transparent are enabling microscopes to visualize the cellular architecture of whole tissues in 3D with unprecedented... tract or CST traversing pons and medulla, bp= basilar pons, NST nucleus of the solitary tract.  The arrows...light using a different objective lens oriented perpendicular to the plane of illumination (i.e. light sheet...multiple-view reconstruction (5). In order to image large tissue such as cleared rodent brain, the Ultramicroscope...was developed using a stereomicroscope to capture large field-of-views (6). For more comprehensive reviews...

...some downsides to working in a large commercial company. In my particular situation, communication between...can sometimes go unnoticed at large organizations. Another downside to large commercial companies which ...cancer research After graduating with a 2:1 BSc in Molecular Biology (roughly a B average in the American system... a type of pediatric kidney cancer. This was particularly interesting as I had just completed my thesis...I changed jobs. I highly recommend taking on a similar position in your field of interest before you dive...apply to study a Master of Science (MSc) in Human Molecular Genetics to give me an edge over other graduates...samples in the lab, but I really struggled with the large scale data analysis, as this is increasingly reliant...

...partitioning machinery away from a larger, low copy plasmid and the larger, low copy plasmid is more likely...plasmids at Addgene Read our Molecular Biology Reference References  del Solar G, Giraldo R, Ruiz-Echevarría...Replication and Control of Circular Bacterial Plasmids. Microbiology and Molecular Biology Reviews 62(2): ...replication and blocking replication machinery (del Solar, 1998). As the copy number of the plasmid increases... replication in plasmids that contain them (del Solar et al., 1998). There are two models for iteron-based...machinery when plasmid copy number is high (del Solar et al., 1998).  In cases of compatible plasmids,...plasmids, incompatibility due to partitioning is similar to incompatibility due to having the same replication...

...involve cellular dysregulation) is the mitochondria. These organelles have their own circular genomes ...been clinically exploited for antiviral purposes similar to that of vaccines. If you know the sequence of... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such as...While these molecules are clearly associated with cellular damage and dysfunction, it is unclear what the...result of bidirectional transcription of their circularized DNA. These dsRNAs are typically contained within... acid hybrids are always around. R-loops are particularly enriched in highly transcribed regions and repetitive...described above play distinct roles in promoting cellular processes and functions, but they also have a ...

...Investigations in Molecular Cell Biology Website. Many thanks to our guest blogger, Clare O'Connor! Clare O'Connor...biochemistry, molecular cell biology and genetics at Boston College. She is particularly interested in...This post was contributed by guest blogger Clare O'Connor an Associate Professor at Boston College. National...computational methods to identify genes by their similarities to genes in other species, but these studies...gene function wide open. What if two seemingly similar proteins have acquired divergent functions due ...strains are available. Then, staff or students clone similar sequences from a less-well studied organism into...confident that the gene from the test species has a similar function to the gene in the reference species. ...

...steps for lysing cells and removing cellular debris look very similar. However, keep in mind that kit components... model, or overall throughput needed. Large constructs – Large plasmids, cosmids, or BACs, can be tricky...from bacterial cultures is a core protocol in molecular biology. And while kit-free plasmid purification...quality preps, while other applications require large amounts of high quality DNA. Figure 1 illustrates... mammalian cell transfections can require quite large amounts of DNA (easily in the micrograms scale and...your plasmid; separate plasmid DNA from unwanted cellular “debris”; and then clean-up the plasmid DNA (Figure... Figure 2: Plasmid isolation workflows are similar across kits. Lyse your bacteria to release the ...

...on a particular DNA-sequence configuration (state) based on the identity and order of a particular set ...mutations, and environmental conditions drive certain cellular behaviors, but little is known about the order... may be used to better understand and engineer cellular processes that are influenced by temporally ordered...input to an RSM drives expression of a distinct large serine recombinase that recognizes its own attB ...reactions are irreversible. Once expression of a particular recombinase is triggered, its effects on the ...registers will likely not be homogenous for one state, regular PCR may not be very informative – each primer pair...fraction of registers that are amplified by a particular primer pair. Then, by leveraging qPCR data from...

...from the cellular debris. There are several popular DNA vectors that can be used to produce large amounts...The central dogma in molecular biology is DNA→RNA→Protein. To synthesize a particular protein DNA must first... post-translational modifications Learn more molecular biology in our Plasmids 101 eBook! Researchers...functions. Proteins can also be purified for use in molecular biology research (eg. polymerases and other enzymes...expression systems can be scaled up for production of large amounts of protein. Some drawbacks of insect cell...insect cells require demanding culture conditions similar to mammalian expression systems. Bacterial expression... answer. E. coli is definitely one of the most popular hosts for protein expression with several strains...

...one example, modular constructs generated with MXS-chaining were used to label subcellular structures in...assembling these modular types of constructs, and different methods are suited for particular downstream applications...enzyme-based). Well suited for the modular assembly of highly similar or repetitive sequences. Intermediate...structure and protein localization, as well as cellular processes like gene expression and cell migration...the fluorophore, and thus enabled labeling of particular structures (Table 1). Each construct was flanked...detection of each fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave...kb insert. Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation...

...shape. These AAV particles are often found with cellular exosomes, small membrane vesicles or microvesicles... - 150 nm diameter) that are secreted from the cellular membrane into the culture medium. AAV particles...inside the vesicle, which may also contain other cellular molecules such as proteins or nucleic acids. This...infect more cells (as demonstrated in different cellular environments such as retina or glioblastoma). ...to neutralizing anti-AAV antibodies. This is particularly useful for therapeutic applications in vivo ...vesicles secreted from producer cells also contain cellular macromolecules (e.g. proteins, nucleic acids) ...retina after intravitreal injection to transduce bipolar cells and photoreceptors (Wassmer et al., 2017)...

... of most molecular biology labs. Recent and rapid advances in technologies that permit large-scale creation... and PCR products are the bread and butter of molecular biology labs the world over. Scientists have traditionally...-based sequencing and confirmation of Addgene’s large and growing collection of curated plasmids from ...technique in our partnership with Addgene to sequence large numbers of plasmids. plexWell™ technology overview...sequential “pooled library prep”, in which a relatively large number of samples are subjected to a DNA-barcoding...normalization problem often arises from the fact that large numbers of individual input DNA samples (e.g. a ...measured in time and consumables, can be daunting for large numbers of samples. As mentioned above, one of the...

...Increasingly popular Optogenetics tools use light to alter cell physiology and molecular processes via...generating DNA molecular weight markers The Tan lab at Penn State - set out to “rule” Molecular Biology. Under...redshifted-emission spectrum compared to that of CyOFP1, a large stokes shift, long fluorescence lifetime, and a ...mScarlet variants have shown great performance in cellular functional imaging and in protein fusions. The...PhoCl between an enzyme and an inhibitor domain similarly allows for control of enzymatic activity. In addition...variants The toolbox of fluorescent proteins (FP) for cellular imaging is constantly expanding. Case in point...point - check out the growing list of popular plasmids in Addgene’s FP collection. Just last month, Robert...

...a pathway to inhibit to block the cellular response to a particular ligand (such as when a component of... are involved in a studied cellular behavior. This approach is particularly suitable for in vivo screens...Institutet, in Sweden. He has a particular interest in using molecular tools to study the immune system...leader of a research group at the Centrum for Molecular Medicine (CMM), Department of Medicine, Solna,...attended a great talk when I was a PhD student. In particular, one slide of the presentation made a lasting...screen and the appropriate phenotypic readout, similar mapping could be rapidly performed by targeting...signaling pathway. For example, one could study the cellular response to a cytokine known to cause the up-regulation...