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...binding partner avidin are commonly used today in molecular biology for an array of different techniques and... interaction and the uses of biotinylation in molecular biology! Learn about in vivo biotinylation of ...biotin is most commonly known for its role in molecular biology, biotin (Vitamin H or Vitamin B7) is first...carboxyl groups to organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier...temperature or organic solvents. Uses of biotin in molecular biology Biotin is a relatively small, water soluble...biotin and its strong interaction with avidin for molecular biology and biotechnology purposes.  Once a polypeptide... or co-expressed with avidin or a structurally similar protein that was discovered in the 1960s, strepavidin...

...this post: Anti-FLAG Frankenbodies for modular imaging of cellular targets Optimized genetic screening technique...editing Anti-FLAG Frankenbodies for modular imaging of cellular targets by: Mike Lacy Adding to their... a set of anti-FLAG frankenbodies, a flexible, modular tool for labeling. Here, Liu et al. engineer a ... cells, Western blotting, and more. Given the popularity of FLAG and HA tags, you may not even need to...single expression cassette is advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a ...create a genome-wide library capable of performing similarly to Cas9 screens in a compacted library. Overall...genome regulation and editing by: Rachel Leeson The large size of CRISPR-Cas effectors and fusion proteins...

...lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. DNA targeting Cas...genome edits, but it is effective at wiping out large regions of DNA and has anti-viral application potential...facts – PAM requirement: AAG or TTC  Best for: very large deletions.   Cas12a – The staggered cutter  Originally...Cas12a cleavage results in 5 bp overhangs and is particularly convenient for targeting experiments requiring... even a single mismatch in the target sequence Similar to Cas12, it is also significantly smaller in size... size than traditional Cas9. Another similarity to Cas12a is that Cas14 also indiscriminately cleaves ...it targets RNA instead of DNA. This makes it particularly useful in generating transient edits of signaling...

...McKenna, Ph.D., is Associate Professor of Molecular and Cellular Biology at Baylor College of Medicine and...Professor and Chair of the Department of Molecular and Cellular Biology, and Editor-In-Chief of the Open...understanding of how these processes are regulated at the cellular, tissue and organ level by nuclear receptors (... receptors and their ligands NRs represent the largest family of transcription factors found in metazoans... organs because of their established roles as molecular sensors and governors of endocrine-hormone signaling..., including hypertension, cancer, diabetes, cardiovascular disease, cholesterol gallstone disease, senescent...provided clues to their previously uncharacterized cellular roles. In the current phase of NURSA we are seeking...

...their detection can suffer from photobleaching and cellular autofluorescence (Tsien, 1998). What sets HiBiT...activity is compatible with both intra- and extracellular environments, making it effective for analyzing...from the Promega Plasmid Collection that express similar constructs available at Addgene. Read the original...HiBiT-TP53 Fusion Vector at Addgene! Following extracellular RNA editors Tracking factors that utilize exosomal... hypothesized that if ADAR1 were carried by extracellular vesicles (EVs), edited transcripts could endow...endoplasmic reticulum quality control navigate a large and interconnected system of dynamic and redundant... endocytic trafficking in real time through extracellular-specific and full lytic measurements. Find CFTR-HiBiT...

...the new fragment thereby generating a complete, circular plasmid that can be easily maintained in a variety...companies now sell fast digest enzymes that can digest large amounts of DNA in as little as 10 minutes, but check...will need to use a phosphatase to prevent re-circularization of the backbone plasmid (see below). You should..., the DNA migrates toward the anode (+ charge). Larger fragments of linearized DNA migrate slower than... to anneal to one another and form a complete, circular plasmid. You then add DNA ligase to covalently...in the transformation due to background re-circularization and contamination from uncut plasmid. 4. Transformation...Additionally, if your final product is going to be very large (>10kb) you might want to use electro-competent ...

...the Liu lab discovered that cellular mismatch repair contributed to a large number of unintended prime ...stability Traditional sgRNAs are protected from cellular degradation by Cas9 binding, but pegRNAs have ...pegRNAs, but combining PE7 with epegRNAs resulted in similar or reduced editing efficiencies, likely because...edits to opposite strands of the DNA and skip the cellular mismatch repair step (Anzalone et al., 2022) PASSIGE...combine prime editing with a recombinase to install large sequences (up to 10 kb) into the genome (Pandey ...deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nature Biotechnology...Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22...

...are not limited to, ones targeting muscular dystrophy, cardiovascular disease, inflammatory diseases, blindness...TTR is primarily made in the liver. Similarly, for cardiovascular disease approaches targeting cholesterol...et al). CRISPR engineering of CAR-T cells is particularly promising because of its potential for allogenic... polymorphisms (SNPs) in the TTR gene and is molecularly characterized by misfolding of the TTR protein...mutations or introducing a desired edit, but this particular class of treatments has quite the opposite aim...therapy currently in clinical trials is somewhat similar (Hussein et al). It employs Cas9 to cut at two ...two sites within the viral genome to excise a large portion of it. This ongoing trial is sponsored by Excision...

...receptors The receptor you choose is largely dependent on what cellular response you aim to manipulate. It...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...normal brain function. Chemogenetics has become a popular tool in the field as it provides a noninvasive ... where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted. Targeting...nonnative ligands. However, they retain their intracellular interactions and still set off the same pathways...been derived from muscarinic receptors. The most popular agonist for muscarinic receptor derived DREADDs...such as the serotonin 3 receptor, will promote depolarization in neurons. Alternatively, combining with the...

...Viral delivery of TnpB for plant genome editing LARRYv2 pooled libraries for cell barcoding Addgene’s own...deposited a variety of constructs for visualizing cellular components (nucleus, endosomes, lysosome, golgi...found itself in the spotlight. TnpBs are gaining popularity, especially among plant biologists, due to their... (learn more in our recent blog post on TnpB). Similar to CRISPR-Cas, TnpB is directed to genomic targets...virus, to deliver a TnpB (named ISYmu1) to the popular plant model Arabidopsis (Weiss et al., 2025). By.../s41477-025-01989-9.   Make a splash with new LARRY barcoding libraries By Mike Lacy Whether you’re ...method is their updated Lineage and RNA Recovery (LARRYv2) barcoding libraries. The team used these libraries...

.... An accidental release would be particularly dangerous for molecular biology and genetics because the...such as malaria and dengue for many years. Since more than a thousand children died of malaria today, ...safeguards and confinement strategies are available? Molecular confinement involves building gene drives that... tropical mosquito vectors of diseases such as malaria and dengue are currently being performed in regions...performed with two or more confinement methods. Since molecular, ecological, and reproductive confinement typically...cost of an accidental release. Science relies on popular support, which in turn depends on widespread trust...shared environment, they must never be used without popular support2. An accidental release could delay real-world...

... as to make plants, and agricultural crops in particular, resistant to disease. They also devise biosensors...biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens...biology Addgene: Tell me about the Golden Gate Modular Cloning (MoClo) Kit. What does it include? Patron...to show that they work well in plants and give similar stability to transcripts as the ones that people...we are collecting sequences that will provide a similar resource for those involved in monocot engineering...workhorse. Tips & tricks Addgene: Do you have any particular tips for those looking to get started with MoClo... parts. That was the motivation for releasing a large parts kit. Once you have so many parts available...

...Resources on Addgene.org Basic Molecular Biology Protocols Protocol Videos Molecular Biology Reference Pages  ...T4 DNA ligase) are critical reagents for many molecular biology protocols, including vector-insert joining...sequencing (NGS) library construction, and circularization of dsDNA (6). Less commonly utilized in vitro...are sensitive to distortions in helix shape (22). Large purine:purine mismatches and most smaller pyrimidine...with products identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method ...novel 16S rRNA gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology.../LDR/flow-through hybridization assay using a capillary tube, probe DNA-immobilized magnetic beads and...

... etc. Contamination by humans accounts for the largest source among those mentioned above. The contaminants...and then specific enzymes are used which trigger cellular mechanisms that generate luminescence. This can... in the form of small clusters, apart from the cellular DNA. Fluorescent DNA staining is another DNA staining...mycoplasmas, contamination by other bacteria or extracellular fluorescent signals produced by these bacteria...masks. Make sure to change the gloves and masks regularly instead of keeping them in the pockets of your...preventing mycoplasma growth. Also, prolonged use of a particular antibiotic can induce resistance for other bacteria...cross-contamination. CO2 incubators should be cleaned regularly and dampness of any kind should be avoided. Distilled...

...lentivirus). Large cargos, such as the ~11 kB cDNA for dystrophin, the gene mutated in muscular dystrophy... being inserted into new target sites. This is similar to how retroviruses, like HIV, replicate. Class...can be inserted between the transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a...vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive version ...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase...with a maximum cargo capacity of ~200 kB of DNA. Similar to piggyBac, Tol2 also prefers to integrate at ...transposons Now that you’ve learned about some of the popular transposon systems, let’s take a look at how they...

...colonies that are comprised of cells with a large nucleus, large nucleoli, and little cytoplasm. Positive...oncogenes, such as Myc. Viral vectors also have the largest genomic footprint of iPSC generating methods due...infect dividing and nondividing cells and have a larger cloning capacity (8-10 kb) than other retroviral...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...transfection efficiency is cell-type dependent and larger plasmids have lower rates of transfection. Minicircles...other methods. This could be a reflection of the large size of the plasmid or silencing of the plasmid ...modifications are used to protect the RNAs from cellular defenses against ssRNA: phosphatase treatment,...

... Laboratory of Molecular Biology. Plasmid cloning is an essential part of any molecular biology project...the MRC Laboratory of Molecular Biology. He is currently investigating the molecular biology of synaptic...for plasmid cloning Find our molecular biology reference here Find molecular biology protocols here  ...current cloning techniques involve the assembly of a circular plasmid in vitro, before transforming it into ...transformed into E. coli and plasmid assembly (re-circularization) occurs in vivo. We use XL-10 Gold E. coli,...Javier García-Nafría from the MRC Laboratory of Molecular Biology. Jake Watson is a postdoctoral researcher...investigator scientist at the MRC Laboratory of Molecular Biology. He is interested in neuronal signal transduction...

...guest blogger Pooran Dewari, a postdoc in Steve Pollard’s lab at the MRC Centre for Regenerative Medicine...workhorses of the cell – never work alone in the cellular milieu. It is, therefore, critical to understand...biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the...spectrometry to identify the interacting proteins. Similarly, in ChIP-seq assays, chromatin fragments that ...ambitious projects that require epitope tagging of a large cohort of genes (e.g. tagging genes representing...success of the epitope tagging experiment depends largely on how efficiently these components are delivered... TAG-IN bioinformatics tool.A recent article by Clarke et al shows that crRNAs binding to the template...

...fingertips the tools to probe cellular dynamics and function with extraordinary molecular detail.   References...offer scientists a simple yet powerful way to tag cellular proteins and investigate protein localization,...fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red fluorescent...photoactivatable fluorescent protein for your experiment Similar to performing experiments with standard FPs, before... chimeric protein will oligomerize, disrupting cellular function and localization, or leading to protein...to optimize your activation protocol for your particular set up. Applications of photoactivatable fluorescent...live imaging of proteins, organelles, and cells. Similar to FPs, PA-FPs allow for non-invasive labelling...

...cells. They bind to proteins on the surface of extracellular pathogens such as parasites or microbes, or ...(Cruse et al., 2004). Because proteins are much larger than this, it may contain many epitopes that antibodies...antibodies are less likely to cross-react with other, similar proteins due to recognizing a single epitope of...monoclonal antibodies here. Recombinant antibodies Similar to monoclonal antibodies, recombinant antibodies...proteins. The proteins are separated based on molecular weight by SDS-PAGE, and are subsequently transferred...internal control for Western blots, shows that a similar concentration of cell lysate was added to each ...   Enzyme-linked immunosorbent assay (ELISA) Similarly, ELISA is used to detect the presence of a single...