...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest... “ligand” that binds to the tag. Like most good ideas in biological imaging, the initial breakthrough ...breakthrough for in-cell labeling techniques was provided by Roger Tsien, who showed that the selective interaction...short genetically encoded tetracysteine (Cys4) peptide tag could be used to label proteins in cells (Figure...tags (e.g., SNAP-tag, HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme...covalent attachment of a fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine...fluorophores were designed as antibody and oligonucleotide labels for fixed-cell imaging, not for live-cell...

...the cell might consider junk protein. Though there are a number of chemical and peptide-based proteosome... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...these challenges, researchers have amassed a considerable molecular tool box of tags and fusion proteins...proteins, such as Thioredoxin A (TrxA) that improve disulfide bond formation in order to help keep your protein...bacterially derived in-vivo biotinylation signaling peptide (Bio), flanked by hexahistidine motifs (6xHis) ... a combo tag comprised of a calmodulin binding peptide (CBP), a TEV cleavage site (more on that in a moment...The efficiency of this cleavage depends on the identity of X. A number of other proteases are available...

...and dampness of any kind should be avoided. Distilled water provided to maintain the humidity should be...contamination (Uphoff, 2002). Mycoplasmas are considered to be one of the simplest and smallest bacteria...Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting mycoplasmas...steps start from you. If you are careful enough to provide a sterile environment for work, it will soon become... coughing in the lab. The human mouth contains a wide variety of mycoplasmas and these can enter the cell...you keep all the required equipment and reagents inside the laminar airflow before and while you are working...regularly as mycoplasma, bacteria, and fungus can reside in these containers. Clean up any media or culture...

...so good with video calls, how about trying your hand at a video presentation? Most video platforms make...months past a worldwide shift to work-from-home and just now realizing that we can’t bide our time and ...are moving forward. Here are a few out-of-the-box ideas that might jump start your learning. 1. Create a...and most of us could use more mentors to listen, guide and support us during training and throughout our...see Form Your Own Peer Mentoring Group: A How-To Guide for Scientists.   Once in-person, peer-mentoring...others and get their input as well. Giving a great video presentation is going to be a useful skill for the...learned to play the ukelele...perhaps you get the idea. What will you start? 4. Do informational interviews...

... It can be applied for identification of essential MYC binding sites in a wide range of human cells.  ...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...disease-causing point mutations. They allow single-nucleotide modifications to edit DNA without requiring a...recently deposited a set of plasmids that helps identify base edited cell populations without end-point...reporter for editing enrichment”, or XMAS-TREE, to identify base edited cell populations without end-point...should have changed from “TGA” to “TGG.”  After identification of ABE activity, XMAS-TREE can also be used...difficult to modify in the past.   Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells are...

...trying to decide which CRISPR tools are best for your experiment, check out our CRISPR guide or leave ...can be used to modify any desired genomic target provided that (1) the sequence is unique compared to the...Protospacer Adjacent Motif (PAM sequence). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9...For more details on the PAM site, check out this video from IGI!) Synthetic S. pyogenes Cas9s with novel...series of positive selection screens in bacteria to identify mutants of S. pyogenes Cas9 that were able to ...Kleinstiver et al. 2015). From these screens, they identified four novel SpCas9 variants with altered PAM binding...Sontheimer EJ (2018) NmeCas9 is an intrinsically high-fidelity genome-editing platform. Genome Biol 19: . https...

...properly onboarded. Most work environments outside of academia provide a formal training regime and the right...transition. In this installment of the Addgene career guide, we’ll think about what causes this culture shock...enjoyable aspect of their chosen career. You can confidently call yourself a Scientist. You work in a lab....during the lab → office transition? I’ve already provided a few counterpoints to some of the causes of culture...colleagues, we’ve come up with a list of things to consider as you make this transition. Of course, a “desk...these--get started with our Transferable Skills Guide.     Over the last decade, my peers and I have... the first place. Finally, if you still find the idea of not holding a pipette again upsetting, there ...

...experimental work, but with this guide, we hope you feel more confident incorporating them into your research... We strongly recommend following the protocols provided by the depositing lab whenever available. Addgene...library sample, and any other information or scripts provided by the depositing lab for data analysis.  I'm ... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in... the virus–the right amount of virus, that is. Ideally, each cell will be transduced by no more than one...from Addgene? Take a page from our book and put aside an aliquot for titering and optimizing before you... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...

...Salegio et al., 2013). Zingg et al. 2017 also provide evidence that AAV1 and AAV9 are capable of synaptic...particle is taken up on one side of the cell and released again at another side of the cell after intracellular...Conclusion An ideal comprehensive toolset for neuronal tracing and network identification requires both...single synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted ...delivering neuronal tracers AAV vectors have been widely used by neuroscientists for some time due to their...proteins. For a brief overview see Addgene’s AAV guide. AAV particles are relatively small (20 nm) in size... transgene expression. Fluorescent proteins are widely used for tracing and their expression is often ...

...as a newbie reprogrammer, to decide which method to use. This post provides a brief overview of reprogramming... them with peptides that help mediate their transduction, such as the polyarginine peptide described in...variation between iPSC clones. Retroviruses are not considered safe for clinical applications.     Lentiviral...Like retroviruses, lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox... site of excision, this approach is still not considered safe for therapeutic applications. piggyBac (...insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors ... includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

...through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman et al...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...popular tools for genetic screens, enabling genome-wide interrogation of the genes required for cellular...processes. For a successful screen, it is necessary to identify the gRNAs that elicit the phenotype, and this ... the gRNAs responsible for a phenotype can be identified by next-generation sequencing of the genomic ...contributed by Leah Schwiesow A leading cause of death worldwide, cardiovascular disease is driven by atherogenic...ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents (what is commonly called...

...excited to provide ready-to-use CRISPR lentiviral preps to researchers across the globe. Why provide CRISPR...for genes involved in a wide variety of cellular processes. While these are widely requested and incredibly...example, we’ve teamed up with the Broad Institute to provide lentiviruses for delivery of SpCas9 and Cas9 nickase...BRAF and MAP3K4 gRNAs allow you to target these widely studied signalling pathways. Find Validated gRNAs...alleviate some of this labor for our users, we now provide the Brunello (targeting human genes) and Brie (...gRNAs to target around 20,000 individual genes. We provide enough lentivirus for you to both perform an optimization-scale...lentivirus stocks. Our research scientists have provided their standard lentivirus production protocol ...

...scatters sideways to the path that it was initially traveling is called side scatter (SSC) and provides information...their observations. However, while flow cytometry provides analysis at the cellular level, it cannot be used...degree of scatter is captured by detectors and provides useful information about the cell. Light that ... traveling is called forward scatter (FSC) and provides information about the relative size of the cell...reporter on the N- or C-terminus of the protein. One downside of fusion proteins is that the tag can alter the...granulocytes in a plot of forward scatter (FSC) versus side scatter (SSC). Users can gate around their desired... analysis, include a complete set of controls. Ideally, every flow cytometry experiment will have a negative...

...Madison lab created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA...well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.  By using...fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and seamlessly...fluorescent protein for assessing CRISPR/Cas9 activity Guide RNA structure and the site in the genome you are...Sander and Joung, 2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR...-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired ...repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry...

...appropriate for your specific research question. Considerations when choosing your Cas nuclease When choosing...experiments, there are three important factors to consider: site accessibility, specificity, and sensitivity...cleaves a site that is not a perfect match to the guide RNA (gRNA) used to target the genomic locus of interest...interest. As these off-target effects can be genome-wide and, in some cases, difficult to determine, the ... off-target cleavage should be an important consideration during any experiment. Sensitivity Cas9 sensitivity...measured by calculating the on-target activity divided by the off-target activity. Figure 2: Specificity...engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al., 2018). The David Liu...

...himself from the idea of starting a company around engineered bacteriophages. He decided to quit his "safety...Operations of Sample6, for advice on how to take an idea developed during graduate school and turn it into...into a biotech company. Transitioning an idea from academia to a startup Mike Koeris and fellow co-founders...co-founders Tim Lu and others first came up with the idea that would evolve into Sample6 as graduate students...using bacteriophages as bacterial therapeutics. This idea was incorporated into Novophage Therapeutics, Inc...that entrepreneurs must clearly communicate their idea at various levels of complexity and abstraction ...people with diverse backgrounds how amazing their idea is. While communication styles in academia and industry...

...structure of the POI would work best. Ideally, structural information can guide this decision. In our experience... This is an important downside of the technology and should always be considered when interpreting results...that is strong evidence that the fusion behaves well. But this is not conclusive evidence that the fusion... any tendency of GFP to homodimerize should be avoided because it will double the size of the protein....or (ii) designed to form an inert, unstructured peptide and therefore consisted of glycines, serines and...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...al., 2011).  Although structural information may guide design, it is advisable to generate multiple constructs...

...size, the cytometer provides you with two measurements: forward scatter (FSC) and side scatter (SSC). To... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...scattered forward at a low angle (FSC) or to the side at a large angle (SSC). FSC and SSC depend on the...three gates will likely be: the FSC/SSC gate to identify cells, the single cell gate to exclude duplicate...running my flow analysis, I can however clearly identify my lymphocyte population (10.7% of all recorded...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...

...clinical applications could be considered. It is important to consider the question of broader applicability...underwent repair not from the single-stranded oligonucleotide (ssODN) template supplied by Ma et al., but...allele rather than the supplied single-stranded oligonucleotide (ssODN) template. Embryos also underwent editing...failure of the embryos to undergo editing using the provided ssODN template. In fact, this finding led the ...conducting research in embryos should our society decide that germline editing is permissible in cases of...disease prevention. However, it is important to consider that already-tested CRISPR alternatives are applicable...target. In addition, as on-target editing rates vary widely from gRNA to gRNA, it’s unclear where this study...

...week. Here we will provide an inside look at the steps we take to verify the identity and quality of the...plasmids through Addgene is that we provide an added level of confidence in the plasmids we distribute through...the plasmids we make available and provide some advice on the steps you can take to verify your own plasmids... ends to verify that the sequence assembly and identity of the insert are correct. In other cases, we ...glycerol stock to each of 2 96-well microplates (identical copies, see image above). We then select forward...corresponding glycerol plate on dry ice to the sequencing provider. Each sequencing service will have different requirements...requirements for volume and concentration, and not all providers are equipped to perform Sanger sequencing from...