...proteins had to be re-engineered for each gene, making wide-scale gene activation seem next to impossible. The... systems only require changing the programmable guide RNA. Gene activation by dCas9, also referred to ...specific sequences, CRISPR/Cas systems rely on a guide RNA complementary to the sequence of interest. Upon...VP64, a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery...of VP64 per each dCas9, SunTag uses a repeating peptide array to fused with multiple copies of VP64. By...two-component system dependent on gRNA design (SAM) or peptide design (SunTag). This streamlines its delivery,..., Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9–based transcription...

... your video call.  Expect technical difficulties. To try and circumvent this, test out the video conferencing...development, and company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer... homework before the interview. If you are not provided with a list of who will be interviewing you, ask... your audience. Connect your prior work or a new idea to the company’s area of expertise. This has potential...difficulties. Have your presentation on a flash drive and consider bringing your own laptop as a backup, if the company...the interview, if possible. Otherwise, read the video conferencing instructions you received from HR carefully...after completing each section of your talk. Some video conferencing software allows participants to “raise...

... the last year for diagnosing COVID-19, aside from the widely used qPCR and antigen tests.  Labs including...Konermann S, Hsu PD (2021) Genome-wide, bidirectional CRISPR screens identify mucins as critical host factors...spike protein was developed based on 100 structure-guided spike designs and has improved protein yields and...During the 2003 SARS outbreak, Hyeryun Choe’s lab identified that ACE2 is the functional receptor for SARS-CoV..., and more. Recently, scientists used the genome-wide CRISPR libraries, Brunello (knock out) and Calabrese...expressing SARS-CoV-2 open reading frames and identified interactions of these proteins with human proteins...Months later, we partnered with Ginkgo Bioworks to provide synthesized SARS-CoV-2 expression plasmids available...

...just isn’t always ideal. But the increased flexibility helps even when it’s not an ideal site: moving several... that you hope is present next to the "perfect" guide sequence for your genome engineering experiment....editing landscape. The need for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality...sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance...flexibility in gRNA selection and subsequent editing. Identifying PAM-free and -flexible Cas enzymes   The solution...more pan-genome PAM flexibility, you’ll want to consider one of several Cas9s that recognize NG PAMs. The...sought to address the PAM problem through structure-guided engineering of the PAM-interacting domain of Cas9...

...genome-wide library deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ... can both process crRNA and carry out the crRNA-guided DNA cutting, unlike other well-characterized Cas...to delivery constraints and scientists seeking a wider range of targetable genomic sequences could benefit...researchers sift through thousands of cells to identify cells exhibiting properties of interest. However...of Yellow Fluorescent Protein (YFP). The team identified a new YFP variant, mGold. mGold was found to ...reduction in performance compared to larger genome-wide libraries. Find the library here New from the...

...example at an anniversary or for Girls’ Day, offer guided tours to show people where the lab magic is happening...share your knowledge at local schools. Or work as a guide in local museums, zoos or botanical gardens. You...Addgene are open to guest contributions. If you decide to start your own blog, check out these two excellent...record a podcast or put some effort into making a video? Use your unique talent to communicate science. ... Guardian, BBC Breakfast or the Londonist. BBC provides a peak into their day-to-day business with popular...Co-Ordinating Centre for Public Engagement (NCCPE) provides on-site courses in public engagement for institutions...media skills, writing about your research and residential communication. If science journalism is the path...

...lentiviruses in parallel, this approach should be considered. Similar to the p24 assay, titering via measurement...When using qPCR based methods for viral titration consider using a reverse transcriptase mutant in a separate...carryover. Functional titer Functional titer is considered a more accurate method to quantify virus as it...procedure can take several months but should be considered in labs that will routinely be producing virus...virus or require precise titers. Additional considerations for titering lentivirus Volume and transduction...batch of virus for long term use, you may want to consider using an aliquot that has been subjected to a ... labs that routinely produce virus may want to consider the more accurate proviral integration assay. ...

...person is able to communicate their ideas confidently while also considering the needs of other people. Some...Bramson, author “Coping with Difficult People,” identified seven types of difficult people: Hostile-aggressives...postdoctoral fellows are hesitant about expressing their ideas, especially if their supervisors are difficult people...frustration or anger, because your discussion will get side-tracked, and you might create even more conflicts...Your supervisor might not agree with all of your ideas, but he or she probably has reasons for it. Do not... strategies will help you to communicate with confidence earn respect from your supervisor. Whenever possible... resolve most conflicts if you communicate your ideas assertively, listen to his or her viewpoint, and...

... asking the panelists not to use any slides for the event. Slides are for presentations, not discussions... the prep calls, but often the prep calls provide good ideas for interesting questions. Be ready to encourage... panelist can contribute. The moderator should identify the funny, provocative or interesting stories ...the actual event, based on these interviews. The idea is to ask questions in a directed way to bring out...discussions. I will rarely work with a panel if slides are allowed. Don't put your panelists behind a table. ...it when the moderator sits in the middle, but the side can work. Only put out enough seats for the expected...more great tips on running panel discussions see Guidelines for Great Panels by Scott Kirsner and How to ...

...instead of recognizing nucleotide triplets, the components recognize individual nucleotides. This imparts TALENs...movie Ethan Hawke’s character revolts against class divide that is created by genetics.    Zinc finger nucleases...repair template.  Each zinc finger recognizes a nucleotide triplet and when several ZFNs are strung together...using a protein to recognize DNA sequences is not ideal. For the early generation gene editors, new amino... protein-based DNA-binding domain with a single-guide RNA sequence.  The Cas proteins, just like its predecessor...prospects of curing diseases caused due to single nucleotide variations in the genome.    Due to the rise ...reverse transcriptase. Using an elongated template + guide RNA called pegRNA (prime editing RNA), the prime...

...procedure The procedure (illustrated in the YouTube video below) is simple: After obtaining antibiotic-resistant...form a firm monolayer. The cell clones - easily identified from the plate circling- are then individually...plug by rapid pipetting, as shown in the YouTube video. Before returning the plate to the incubator, additional... a part of the body very sensitive to heat- the inside of the wrist or the cheek. (Parents of infants ... well separated. For this, you need to have some idea of the rate of growth of your specific cell line...cells we sometimes use TPP peelaway T-75 flasks to provide a greater surface area (the amount of agarose/DMEM...transfected clones. The technique is applicable to a wide variety of cell lines, from HEK to CHO cells to ...

...polymerase, tRNAs, ribonucleotides and amino acids. Cell-free expression systems are ideal for fast assembly... can then be translated at the ribosome into polypeptide chains that make up the primary structure of ...modifications including protein folding, formation of disulfide bridges, glycosylation and acetylation to create...Mammalian expression systems Mammalian cells are an ideal system for the expression of mammalian proteins ...of the T7 lac promoter and induced by lactose, provides the highest level of protein expression. Despite... and patience. Plant expression systems Plants provide a cheap and low-tech means of mass expression of...recombinant proteins with a naturally secreted plant peptide allows for easier access and purification of a ...

... human gut flora. Guides targeted essential and non-essential genes with two guides per gene. The library...expressing guides that target essential genes were less represented while bacteria containing guides targeting... Jason Peters, Oren Rosenberg, and Carol Gross provides a streamlined suite of tools for using CRISPRi...Higher transfer rates are better suited for genome-wide CRISPRi library screening experiments where maintaining...biology of bacteria, Mobile-CRISPRi can be used to identify antibiotic resistance genes. Genes that are targets... modular nature allows for the customization of guides, promoters, selection markers, and different versions... Find bacterial CRISPR plasmids Read our CRISPR guide ...

...need some help. Today in our transferable skills guide: Negotiation. As a grad student, I thought that...perspective of all persons involved and be used as a guide to point you toward a creative solution where everyone...specific details about the individuals, each example provides a scenario where both people get something valuable...example shows, the ultimate resolution can involve ideas not related to the actual data collection task but... relationships with your colleagues within and outside of your organization. In my non-academic roles,...investing in me is also beneficial to them? I have an idea on how to improve my current workflow but I need...negotiation  Skills Snapshot: Negotiations When Cultures Collide: Managing Successfully Across Cultures by Richard...

... of their undergraduate experiences, they were provided with ample resources and a positive perspective...In 2018, the ASBMB and Burroughs Wellcome Fund provided seed funding to take this initiative to the next...just released report “Enhancing Dissemination of Evidence-Based Models for STEM PhD Career Development; ... for pd|hub which include building capacity to identify the most effective practices and disseminating...training approaches in a way that can be implemented widely in the community.  Talk alone is not going to drive...development and implementation of collections of evidence-based practices. These concrete programs and practices...PhD professional development are intended to be widely disseminated by “train the trainer” programs.  ...

...fluorescent dye or HRP via maleimide-labeling. Maleimide-labeling occurs when a maleimide group forms a covalent...found in cysteine. Maleimide-conjugated HRP can be found here and several maleimide-conjugated dyes are...reporter enzyme. For multicolor staining, careful consideration must be used when selecting which antibodies...only one primary antibody. This process can be avoided direct antibody fluorophore conjugates are used..., you can learn more about how nanobodies are identified in this review. If you just want to use the nanobodies...nanobodies are outlined below; no alpaca required. Decide Which Nanobody To Use: The table below summarizes...

...minimum, two components: the Cas9 protein and a guide RNA (gRNA) targeting your genomic site of interest...are some potential complications that must be considered when using this method:     Cells must be amenable...: generating single or multi-gene knockouts in a wide variety of cell types, gene editing using homology...of plasmid or viral-mediated delivery. You’ve decided to use RNPs, now what? Preparing the Cas9-gRNA ...native C547, allowing for ligation to pyridyl disulfide-activated ligands. Due to the concerns of off-target...is most appropriate for a given experiment. Considerations for RNP-mediated CRISPR experiments While using...with these types of experiments. One important consideration is that the Cas9-gRNA RNP will be degraded by...

...unturned, but cancer has been producing landslide after landslide, only adding more stones to the field.... recognition are a few methods by which tumors sidestep immune surveillance: Suppressing Function Antigen...beyond direct proximity to the tumor; chemokine-aided activation of APCs produces modified dendritic cells...However, these conventional therapies produce lasting side effects, damage healthy cells, and often cannot ...tumors. Monoclonal antibodies have been shown to aide chemotherapy and result in successful elimination...include: Cell heterogeneity Tumors are composed of a wide variety of cell types. This cell heterogeneity is...Tumors differ in their genotypic and phenotypic identities based on their level of progression, anatomical...

...with your colleagues. Start small by identifying the protocols, guides, or references that your labmates .... Or maybe you can’t find the molecular cloning guide you were sure was in one of those four white binders... the spine. For binders of protocols and data, consider using color coded binders and tabs (for different...easier to know where to start your search. Then identify the most frequently used pages in your notebooks...to protocols in real time. You may also want to consider moving your paper spreadsheets for reagent storage...information is backed up and edit tracking will help you identify when others have added or removed samples. Once...worked for you. Share your best tips, tricks, and ideas below. ...

...transparent zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis...similar processes occur over days inside the mother, the zebrafish is an ideal system to observe embryogenesis...zebrafish-specific gene expression databases that provide information about when and where genes are active...tools for zebrafish Random mutagenesis screens provided a major toehold into understanding how genes control...deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. When injected into zygotes...applications and conclusions Zebrafish are also popular outside of developmental biology, and are particularly ... the biological question, and zebrafish are not ideal for every application. Disadvantages include their...

... sgRNAs. Its RNA-guided nature makes CRISPRa much simpler to implement in genome-wide screens over cDNA...components, and several sgRNAs must be screened to identify effective ones. Simplified in vivo delivery using... then isolated and their genomic DNA sequenced. Guide RNAs targeting several known and some unknown regulators...including microglia, astrocytes, and oligodendrocytes, provided that reporter genes specific to these cell types...of good flow cytometric antibodies for many CNS-resident cell types (microglia are a notable exception)...are often many more genetic strategies) and then consider whether traditional cDNA overexpression is suited... al. "CRISPR activation screens systematically identify factors that drive neuronal fate and reprogramming...

...always easy to identify the ideal codon for every amino acid across an entire polypeptide. Many researchers... growing polypeptide chain. One part of the tRNA contains the anticodon,  a three nucleotide sequence ...overview of peptide synthesis. A ribosome interacts with mRNA and charged tRNAs to build a peptide. Image ...Usage Tabulated from GenBank database (CUTG), that provides an electronic dataset for codon-usage of 257,468...synonymous codons based on an organism's codon bias. The idea is that scientists can make mutations throughout...

...needed to know more about. I caught up with her outside the room and sheepishly explained my situation ...established their own labs.  Fried chicken with a side of inspiration  I look back at the meeting with ...thought that binning Singletons together would provide a structure and support network for people attending...attending large meetings. A key was to provide each bin with a mentor who had attended the meeting multiple...promotion from those who could not participate but provided moral support was invaluable.  Mixing metaphors... comfortable at a large meeting. They had the confidence to meet someone new, as they’d been introduced...of approaches scientific meetings have taken to provide mentoring. For example, Goldschmidt conference,...

...). Genome-Wide Recessive Genetic Screening in Mammalian Cells with a Lentiviral CRISPR-Guide RNA Library..., targets 18,080 human genes with 64,751 unique guide sequences to enable both negative and positive selection...including Feng Zhang used the GeCKO library to identify genes essential for cell viability in cancer and...David Sabatini and Eric Lander, comprise 73,171 guide RNAs (gRNAs) covering a total of 7,114 human genes...target a smaller set of genes and it includes more guides per gene. The Sabatini/Lander library uses sequential...uses a 1 plasmid, single infection system. Genome wide mouse CRISPR library The Yusa lab has leveraged ...genes involved in bacterial toxin resistance, identifying 4 previously unknown genes.  “With this work,...

...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...approaches and organoid models, toolkits like this one provide new opportunities for investigating genetic mechanisms...2020.05.04.076067    Novel fluorescent enzyme biosensors provide direct, specific and sensitive detection of the... rather than on the fluorescence intensity, it provides a direct and quantitative detection of SLs. The...plants.  FnCas12a, unlike other Cas12a nucleases identified, functions at temperatures up to 43 °C and can... on NGG PAMs relative to wild type. A new genome-wide CRISPR knockout library from Xiaole Shirley Liu'...targets all mouse genes with 10 gRNAs per gene. The guides are optimized to maximize on-target cleavage and...

... cell lines used in scientific research are misidentified! Rigor and reproducibility of scientific data... cell lines. Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research, ...and collections. Importantly, Cellosaurus also identifies problematic cell lines: ones either suspected...or easily contaminated, as well as commonly misidentified lines. Browsing this feature to know if your...the known features and relevant literature, or to decide which line is best for your experiments. You can...and shared allele search preferences to help you identify proximal lines you care about. If you have cell...is as good a time as ever to find out the true identity of the line? Once you’re run your STR profiling...

...Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide Blocks 50S...Resistance to antibiotics is a widely used tool in molecular biology, yet scientists rarely stop to think...when the cells are grown on selective media, and provides those bacteria with a pressure to keep your plasmid...Action*   Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; causes mis-translation...mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit; interrupts protein... Bactericidal 7.5-50 ug/mL Streptomycin aminoglycoside Inhibits initiation of protein synthesis ...Bacteriostatic 50-100 ug/mL in EtOH Polymyxin B polypeptide Alters outer membrane permeability Bactericidal...

...others. Another peptide-based strategy uses an enzyme to covalently label the peptide with a fluorophore... Balla. "Live cell imaging of phosphoinositides with expressed inositide binding protein domains." Methods...University of Amsterdam). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several...proteins as described above. The smallest tags are peptides that are engineered to bind specifically and with...homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production...the continuous effort of numerous scientists world-wide.   Many thanks to our guest blogger, Joachim Goedhart...PubMed PMID: 10748019.  8. Lotze, Jonathan, et al. "Peptide-tags for site-specific protein labelling in vitro...

...in bacteria, so many that it may be difficult to decide which path to take. A quick and easy way to introduce... the Lambda bacteriophage, is arguably the most widely and productively used  (Thomason et al., 2014)....In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless mutations, both in stand-alone...What are the counter-selectable markers used to identify double crossovers? The most commonly used counter-selection...the genome. Subsequent selection on sucrose will identify double crossovers. This can be an efficient method...unmarked mutants, but it frequently requires considerable optimization to ensure strong counter-selection...Central PMCID: PMC4862005. Jiang, Wenyan, et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems...

...or developmental characteristics that make them ideal for studying in a laboratory environment. We’ve ...manipulation and genetics research. Plasmids are ideal tools for studying genetic modification for their... E. coli biotin ligase, BirA, has been used to identify and study protein-protein interactions in proximity...pathological developments (Vieira et al., 2019) Besides their ability to regenerate, the axolotl has several...several attractive characteristics that make them an ideal model organism; they are relatively simple to maintain...have large, easily modifiable embryos, which is ideal for observing and manipulating the entire developmental...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...

...proper formation of the disulfide bond between the VL and VH domains. This bond provides stability and solubility...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and... two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long and made...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012...oxidizing periplasm of bacteria to allow for proper disulfide bond formation.  expression in strains of bacteria... concentrations in the eye with minimal systemic side effects. scFvs are part of engineered chimeric antigen...determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable...

...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...while also supporting imaging, making it easy to identify dozens or hundreds of particles surrounding each... A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single ...with MagIC-cryo-EM. Nucleosome-like particles identified by Topaz software (green circles), magnetic bead...its chaperone NPM2, but the technique should be widely applicable to other targets. The modular system..., M., Kamath, N., … & Weissman, J. S. (2024). Brainwide silencing of prion protein by AAV-mediated delivery...Samelson, A. J., … & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based...

...clusters under blue light Check out our Optogenetics Guide to learn more about how these modules work, and ...important components of the cytoskeleton. They provide cells with physical structure, separate chromosomes...microtubules, Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs manipulate ...by Rho family signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner...system (Mahlandt et al., 2023). These Opto-RhoGEFs provide a reversible and non-invasive way to activate a...efficiency in mammalian cells and in vitro, opening up a wide realm of possibilities. The CRY2-SuperPLD module...membrane or lysosomes (the earlier article also provided CIB constructs for targeting Golgi, ER, and endosome...

...spectrometry to identify a mass shift proportional to a single phosphate moiety on the peptide of interest...databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events ...incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...extension and translation of the pSer-incorporated polypeptide. These three components were shown to effectively...mutation. This emphasizes the advantages of bona fide pSer-incorporation into recombinant proteins as ...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...and non-phospho-bands will give the researcher an idea of the purity of pSer-incorporated to non-phosphorylated...

...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...your next AAV experiment, use these questions as a guide for designing your future injection experiments....injection and detection methods. For example, you can provide location, titer dose (Example: 1E13 GC/mL) and ...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...to give proper attribution.  We hope that this provides a clear overview of what you need to submit data...tracing Resources on Addgene.org Read our AAV guide Browse all  AAV vectors Find ready-to-use AAV preps...

....” Subscribers to Scientific America would be considered the interested public and it would be safe to...scientific or science communication. A Tweet is still considered scientific communication if the language is aimed...communication.  Formal education is education that is provided through a structured environment such as the classroom...will.  Informal education is education that is provided, typically ad hoc, for learners who can choose... only used in the field and terminology that is widely used but has a very specific use-case (definition...the context of disease testing, it can still be considered jargon in most other uses. Many laypeople would...communication work does not explicitly exclude people outside of the original recipients. Instead, it targets...

...Park et al., 2019). This provided an engineering road map of sorts, giving an idea of the stiffness the fibers...Resources on Addgene.org Optogenetics guide Chemogenetics guide BRAIN initiative plasmid collection ...cumbersome systems during experiments – neither ideal for the intrepid researcher. There existed, therefore...that could continuously monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes...important as its capabilities are the practical considerations of actually using it - the device has a rechargeable...indeed stable for continuous monitoring. On the gut side of things, the authors optigenetically and chemically...

...make her work understandable to people both inside and outside the scientific community. We’re excited to...making their own fluorescent protein technologies widely available to the research community. To honor their... proteins in which Dronpa domains flanked either side of an enzyme’s active site. In these fusions, ultraviolet...domains could be placed in amino acid loops on either side of an enzyme’s active site and still block enzyme... will be able to use similar designs to create a wide variety of enzymes and proteins with inducible activity...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...

...meeting with no idea why you are there. Make the most of everyone’s time by considering the level of meeting...meetings spent on details that do not yet need to be decided. Figure out what you need to accomplish to get .... Team Meetings: For standing team meetings, provide agenda sections for each person to add a bullet...meetings tend to fill the shape of their container. Consider whether you actually need a full hour to achieve...the time down to an hour or less and are able to provide more timely feedback on each other’s projects. ...each of your meeting types (see above). We’ve provided some tips above for what works well at Addgene...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

...not identical, to their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions...from a colleague down the hall, it is always a good idea to take some time to confirm that you are working...gel correspond to the predicted sizes. Watch this video for a quick overview of how to analyze a restriction...accurately visualize the full size of your construct. Consider buffer and temperature compatibility when digesting...avoid this issue. For your gel: Add ethidium bromide (EtBr) to your gel before pouring it. EtBr binds...your sample settles in the gel well and the dyes provide a visual reference point so you can easily assess...Molecular Biology Reference Find More Protocols and Videos      ...

...even CRISPR based gene editing. But, how do you decide which tools to use, and what do you need to know...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...signaling pathways. Light exposure removes the block provided by the LOV domain allowing the pathway to be activated...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...activate or inhibit a cell in succession and maintain fidelity with the stimulus. For example, if you want to...

... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B). To label...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...interpret. Check out (Jambor et al., 2021) for more guidelines for creating clear and informative figures. Pro-tip...

...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...downstream of the promoter found in the backbone. Ideally, the backbone will contain a variety of restriction...plasmid backbone looks like the one found on the left side of the image below. It has a promoter (blue arrow...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...Methylene Blue Post Visible Light 100 Ethidium Bromide Pre and Post UV Light 0.5 For more information...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...about the company and what the goals were and so I decided to join. I’ve been working with Addgene since 2014...distribution of the plasmids and their ability to identify and fulfill a need in terms of helping the scientific...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...perspective, what do you think are important considerations for the future of open science? What about ...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

...cloning strategy? A screen will help you more easily identify successful clones so you have to weed through ...relies on antibiotic resistance. But what about identifying clones that contain your desired insert? This...ampicillin plates containing X-gal allows you to identify cells that took up the vector and distinguish ...at a few screening methods. Blue-white screen A widely used method of screening is the blue-white screen...example, choose enzymes that only cut once on either side of the expected insert. If the plasmid contains ...planning to go this route, check out our protocol video on using restriction digests to analyze plasmids...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...

...2004, that was a fairly simple goal… and we had no idea how much our understanding of what accessibility...did! From Addgene's very beginning, we had to consider digital accessibility for our website and online... spaces. Alt text is used by screen readers to provide visually impaired visitors with information about...educational content conflicted with plain language guidelines.  Which is why, at the Society for Neuroscience...our workshop how they felt when they read papers outside their field, the answers were primarily negative... Ultracentrifugation First, you’ll want to identify the reader’s goal. Why are they reading this page...and learning how to purify AAV.  Second, you’ll identify your purpose in using that image. Try using the...

...engineering designs and ideas ranged from a biological breathalyzer to pesticide breakdown, production ...engineering of biology. Participating schools are provided with synthetic biology lab kits and lesson plans...recognized name in the field of STEM education worldwide. The BioBuilder curriculum is now taught in almost...and teachers can investigate together. Teachers decide which BioBuilder lessons to teach, and how — some...know-how to design/build/test their own project ideas using synthetic biology.Launching with a kickoff...newsletters, pairs teams with STEM mentors, and provides a nationally recognized platform in March to exhibit...Synthetic Biology Blog Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids for Creating...

...binds to the target DNA sequence as directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats...c-di-GMP in response to far-red light (730 nm). This nucleotide triggers the translocation of the protein BldD...Simply expressing anti-CRISPR proteins does not provide much control over when Cas9 activity is blocked...2020). Fluorescent dCas9 proteins can be used to identify specific genomic loci for imaging techniques. ...architecture. Yu et al., 2020 FAST BphS from R. sphaeroides Split Cas9 Genome editing by NHEJ and HDR. Can...Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018 ...al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...

...protocols Find protocol videos on our website Read our Molecular Biology Guide ...material. Then, the homogenate was loaded onto cesium-chloride gradients and ultracentrifuged for up to 20 hours...However, instead of RNA separation using cesium-chloride gradients, water-saturated phenol, sodium acetate... and variations on this no-kit method are still widely used today (Chomcynski and Sacchi, 2006).  View... tissue collection and homogenization. The most widely known of these products is TRIzol® (also called...Allewell and Sarma, 1974). Additionally, ThermoFisher provides a protocol on how to integrate RNAlater® with ... trick is to precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but...

...single nucleotide polymorphisms. Fast facts – PAM requirement: none. Best for: high fidelity ssDNA targeting...engineering powerhouse, and for good reason. It is guided by an RNA molecule, the gRNA, to a complementary... and an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What ...ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a single mismatch in the target...won’t cut it – literally. Cas13 Cas13 is an RNA-guided nuclease, however, unlike Cas9, it targets RNA ...Abudayyeh, O., Omar, et. al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell...Website CRISPR Plasmids and Resources CRISPR Guide  ...