...using two membranes during your transfer instead of one. If you see signal on both membranes, you know... run the same protein concentration in multiple lanes and cut up the membrane to treat each lane with ...   Figure 2: Reagent gradient. Here, the lanes are lightly marked on the membrane (1) using the... time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...troubleshoot.   Before you start… Mark your membrane! Membranes look the same from both sides, so it’s quite easy...may be under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins...(< 15 kDa) and a 0.45 µm (p) membrane for larger ones. Changing your membrane size is probably the easiest...

...individual clones from that population. You can additionally calculate the minimum number of clones you would... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions... success? Before you start analyzing individual clones, it’s wise to quickly assess whether a significant...verify that you won’t need to screen hundreds of clones looking for your edit of interest. For all of the...before going on to generate and screen individual clones. If a clonal line isn’t your end goal, then a single...is sequencing trace files of WT and edited cell lines. Then decomposition analysis is performed yielding... clone, you should screen, at the very least, 4 clones. Did you use a dual guide system to generate your...

...repression of target genes in plants and many of the aforementioned empty gRNA backbones also contain Cas9...for targeted modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR...be used to knockout, activate or repress target genes in plants using the same general experimental design...cells. Like other model systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as...respective species. Addgene carries >30 “empty gRNA” backbones which contain a plant pol III promoter and gRNA... can also be used to activate or repress target genes in plant cells, respectively. Cas9 expression is..., and a Ti "helper" plasmid, which contains Vir genes necessary for T-DNA processing and insertion into...

... by the heavy-chain constant genes μ and ẟ. These are the first two genes in the heavy-chain constant ...intervening heavy-chain constant domain genes, placing the variable domain genes next to a different constant domain... you heard of an antibody isotype? An isotype determines several key characteristics of an antibody as...an antibody’s isotype is important because it determines what immune cells and molecules are recruited...nomenclature. Antibody heavy chain proteins as well as the genes that encode those proteins are designated by the...tears, saliva, mucus) where it protects mucosal membranes. This isotype is most commonly a dimer with a ...type of DNA recombination. Heavy chain constant genes are clustered together downstream of the variable...

...planarian genes, we encourage you to deposit them back with Addgene and expand the number of genes that other...Hybridization (WISH) for visualizing expression of genes. Double-stranded RNA (dsRNA)-mediated RNA interference...expression and functions of key regeneration-related genes in four inexpensive and commercially available planarians...and Phagocata gracillis. There are 11 different genes cloned into plasmids to investigate for each of ...changes to visualize the expression of specific genes. After riboprobe binding (via hybridization) specific...The expression patterns of 11 different planarian genes (all of which are available through the deposit)...powerful tool for performing targeted knockdowns of genes of interest and was first discovered and characterized...

...computational methods to identify genes by their similarities to genes in other species, but these studies...MET genes to grow.) Students then use replica plating to determine if expression of MET/Met genes from... to answer questions about the evolution of the genes involved in methionine synthesis (5). The budding...strains with overexpression plasmids carrying MET/Met genes from either S. pombe or S. cerevisiae. Students ... the coding sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1...uracil supplementation (6). Expression of MET/Met genes from the pYES2.1 plasmid is controlled by the GAL1...have shown that most, but not all, S. pombe Met genes complement the corresponding S. cerevisiae met deficiencies...

... light include chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent...that, although they are not bioluminescent, some types of corals and anemones are vibrantly colored. He ...Luminescent molecules are very useful tools because we can easily detect and measure the light they emit...Protein (GFP). These molecules occur naturally in bioluminescent organisms, but their real power lies in the...interesting. Although humans have known about bioluminescent organisms and their characteristic radiance..., in the 1960s, Osamu Shimomura studied the bioluminescent properties observed in the crystal jellyfish...the culprits. He even obtained samples of certain ones from local pet shop aquariums in Moscow where he...

...bacteria, transposons can move antibiotic resistance genes between plasmids or from plasmids to the bacteria... and mechanism of replication. They contain two genes: gag and pol. The pol polyprotein encodes the reverse...transposon in vertebrates. It was discovered in Japanese medaka fish because its insertion into the fish... how they can be used in the lab. Transposon mutagenesis screens Transposons, by their nature, are mutagenic... elements, which makes them a great tool for mutagenesis screens that detect loss-of-function or gain-... these screens, the transposons encode reporter genes, mutagenic cassettes, or barcodes. When transposons...to consider when selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best...

...perturbations that may play significant roles in cancer pathogenesis. Their work culminated in the first functional...help cancer researchers studying an assortment of genes. Using patient data to identify mutations of unknown...mutations with strong phenotypic effects can mask weaker ones. Thus, the need for a faster and higher-throughput... team identified 1,049 perturbations in several genes spanning 33 cancer types. The types of mutations...lentiviral constructs using the High-Throughput Mutagenesis and Molecular Barcoding (HiTMMoB) technique (.... Plasmids carrying the corresponding wild-type genes were constructed as well. They called this library.../F3 and MCF10A are growth factor dependent cell lines and any mutations that that confer proliferation...

...Syndrome (a disease that results in combined deafness and blindness) is caused by defects in the gene MYO7a...Jean Bennett started working on ways to deliver genes to correct genetic conditions over 25 years ago ...to the identification of several disease-causing genes, including the RPE65 gene responsible for an autosomal...autosomal recessive, severe, childhood-onset form of blindness name Leber’s Congenital Amaurosis (LCA). Shortly...treatment, famously visited Congress to help increase awareness about the potential of gene therapy!). It would... packaging capacity is well suited for larger transgenes. Unfortunately it has limited tropism in the ...used in clinical trials, a variety of engineered ones have shown great promise in preclinical studies ...

.... Maintaining openness and looseness of spirit The second crucial element in harnessing the power of serendipity...beyond its confines. The beauty of serendipity often emerges at the intersections of disciplines. Cast your... serendipity is maintaining an openness and looseness of spirit. Amidst the rigors of PhD studies and ...routine. It encourages you to think beyond the confines of a predefined path, opening doors to novel ideas...expected. By widening your search and maintaining an openness to unexpected possibilities, you not only invite...

... the paucity of Cre-dependent mouse lines for overexpressing genes within discrete cell populations makes...libraries. However, using CRISPRa to target individual genes can be relatively laborious because multiple plasmids...several sgRNAs must be screened to identify effective ones. Simplified in vivo delivery using SPH mice CRISPRa...neurons over other promoters, including ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus...resulted in hundreds of differentially expressed genes between Bdnf I-overexpressing neurons and Bdnf IV-overexpressing...IV-overexpressing neurons. Among the upregulated genes common between the Bdnf I and Bdnf IV overexpression...overexpression variants were other immediate-early genes associated with neuronal activity, such as Fos, Egr3,...

...Addgene also has ready-made entry clones available for many popular genes, including Hs.KRAS4a. Use our website...selection Used to clone attB-flanked genes of interest to generate entry clones Entry vector attL sites for...competent E. coli cells and select the positive clones. The entry clone and destination vector carry different... recombination step. Addgene’s ready-made entry clones can be used with a large variety of plasmids. Speed...for recombination Used to generate entry clones by TOPO cloning or by Restriction Cloning Destination...the gene of interest in the appropriate system Recombines with the entry clone to generate an expression...plasmids with Addgene. These can be used to express genes in a variety of model organisms. Use the links below...

... for a molecular biologist who wants to express genes from one organism in another? Let’s take a look ...correlated with codon bias across all endogenous genes in E. coli and S. cerevisiae (Tuller et. al., 2010...usage frequency in a reference of highly expressed genes (Sharp and Li, 1987). In 2000 the Ikemura lab created...an electronic dataset for codon-usage of 257,468 genes across 8,792 organisms (Nakamura et al., 2000).  ...2017. Most commonly, scientists codon optimize genes from eukaryotic organisms for expression in prokaryotic...optimizing a human gene for expression in E. coli. Genes are also optimized for expression in mammalian cells... lab optimized the C. elegans GluCl ion channel genes for expression in mammalian cells. This was the ...

...CRISPR-Cas can harness the power of MoClo to expand both targetability and effectiveness. Perhaps the most...and Plant Parts Kit III (Gantner et al., 2018; Chamness et al., 2023). The Plant Parts II kit has a focus...for both monocots and dicots (Hahn et al., 2020; Chamness et al., 2023; Stuttmann et al., 2021; Grützner...CRISPR components expressed from different plasmid backbones. They noted that backbone differences such as ...Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. 2017. Crossref, https://doi.org...Crossref, https://doi.org/10.1021/sb4001504. Gantner, Johannes, et al. “Peripheral Infrastructure Vectors and... https://doi.org/10.1371/journal.pone.0197185. Chamness, James C., et al. “An Extensible Vector Toolkit...

...: rare. “Nature has many genes where there are not rare codons and many genes where there are instances...trend of sourcing genes from distantly related organisms. In 2006, less than 1% of the genes deposited at ...synthetic or natural with 97.7% accuracy. Classifying genes as natural or synthetic But how can you tell if ...in a test set of 173 known synthetic and natural genes.  In designing and optimizing their algorithm, ...synthetic. But nine years later, over 20% of the genes deposited that year were synthetic. They also found... Proteobacteria - the most frequent transfer of genes is from Proteobacteria to mammalian expression vectors...Thompson. "Gene synthesis allows biologists to source genes from farther away in the tree of life." Nature communications...

...experiment. Nucleic acid based machines Recombinase-based State Machines Enable Order-dependent Logic in... cell. These so-called recombinase-based state machines could potentially be used to report upon and/or...post to learn more about recombinase-based state machines and how they function. Adapting Toehold Switches...replication and transcription as well as reporter genes. To learn how you can use minigenomes in your research... describes R-bodies, “force generating protein machines that can extend and retract over many microns ...also showed that R-bodies can rupture E. coli membranes. Read the full post for potential applications...Adeno-associated viruses (AAVs) are commonly used to deliver a genes of interest to a variety of cell types in-vivo. ...

...unstructured peptide and therefore consisted of glycines, serines and other small, non-aliphatic amino acids...fluorescent proteins to oligomerize is known as “stickiness.” Since the fluorescent tag should operate as... per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration...oligomerization in cells. In addition to measuring stickiness, several researchers have also collected data...fluorescent protein characteristics such as the brightness, photostability, maturation time, and acid sensitivity...characteristics are important, but have less priority than stickiness. In my experience, fluorescent proteins that ...mediated HDR) enables the tagging of endogenous genes close to native expression levels. However, it is...

...consider effective brightness of NIR FPs in cells, in addition to their molecular brightness (a product of ...Molecular brightness vs. iRFP713, % Oligomeric state Photo-stabilityc, t1/2, s pKa Brightness in HeLa ...coefficient) (Shcherbakova et al., 2015). The effective brightness reflects the concentration of BV-containing molecules... photoreceptors have higher apparent cellular brightness than previously reported widely used dimeric ...not yield to (m)iRFPs in cellular and molecular brightness (Table 1).  NIR reporters and biosensors NIR ...-the-art NIR FPs, which combine high cellular brightness and monomeric state, are highlighted in blue....Oliinyk, O.S. & Verkhusha, V.V. How to Increase Brightness of Near-Infrared Fluorescent Proteins in Mammalian...

...the secretory pathway. Those can include cytokines and chemokines; enzymes like kinases, phosphatases, and...interchangeably to refer to any technique that combines a marker with a signal. For clarity, Addgene uses...of such targets include transcription factors, histones, and DNA repair enzymes. Note that when using ...label cytosolic proteins alongside the intranuclear ones. You do not need to use a separate fix/perm cytosolic...non-antibody-based stains that bind DNA or free amines of proteins. Depending on the application, they...membrane-impermeant, and the cells of interest can be the ones that are positive or negative for the dye. The main... can only enter cells with compromised plasma membranes — i.e., dead cells — while live cells are protected...