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...” has me considering the microbiome more often than not). If you’re someone who’s considering getting ...definitely enjoyed the lab!) and would therefore consider myself a neuroscience novice (newb). In this post...entire story-telling session organized by Story Collider and which featured a talk about the different ...Developing better AI programs gives neurobiologists new ideas about how the brain functions and vice versa. Even...getting into neuroscience from the mol bio side you’re sure to have many opportunities to learn about and develop...large data sets. Whether these data sets include videos of behavior, many many images of brain regions,...particularly interested in using AAVs because they provide a highly versatile means of delivering genes to...

...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...Now I write for the @Addgene blog & I'm my lab's resident #scicomm expert. What tips do you have for using...discuss how scientists can start using Twitter and provide real-life examples of how they and others have ... February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post from Dr. Paul Knoepfler Hopefully...pknoepfle 3. "Social Media as a Scientist: A Very Quick Guide" from Nature Jobs Blog While Twitter is a popular...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...Addgene blog. 5. "Social Media in Your Working Day" video from Science Media Savvy At this point, you might...

...maintenance within an E. coli cell, but vectors can be widely utilized across many different cell types and each...budding. Yeast Centromere plasmids (YCp): These are considered low copy vectors and incorporate part of an ARS...are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle...most commonly used selection markers in yeast and provides the element needed to overcome the auxotrophy ...as additional uses for said element. This link provides a more extensive list of yeast auxotrophic markers...auxotrophic selection in E. coli? Additional considerations HIS3 L-hisitidine no S. cerevisiae  no yes...experiments. MET15 L-methionine and overproduces hydrosulfide ions yes - Grow with methylmercury. S. cerevisiae...

... approaches a host cell surface are complex polysaccharide structures, many of which are easily varied...proteins could function as surrogate antibodies for identifying and characterizing sugars and sugar-containing... anything else, making it difficult to quickly identify possible gene function. In this post, I will describe...assay, including examples of RBPs successfully identified using this method. Step 1: Prepare a random fragment...transformants for appropriately sized inserts. We consider a library worth using if at least 40% of colonies...overnight), flip the membrane over and place it colony-side up onto a fresh LB plate containing an appropriate...onto a lysis solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After...

...want to reconsider your approach, and try a different cloning strategy. Once you've identified potential...plasmid and all the steps you will have to take. Decide which technique you are going to adopt (i.e. restriction...digestion on an agarose gel. If the digestion does not provide the fragments of the size you expect, check the...region of the primer to 40-60 bp. It is always a good idea to perform primer optimization, especially if you...that, they can be open source (i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays...Now that you've read our plasmid troubleshooting guide, you hopefully have some new strategies to tackle... the lab he develops new assays and processes. Outside the lab, he enjoy the outdoor in all the possible...

...Instead they are detected with tyramide signal amplification. Tyramide signal amplification occurs when...are secreted from cells due to their IgGκ signal peptide sequence, making them easy to harvest from cell...when HRP converts a fluorescently labeled tyramide molecule into a highly reactive radical, which is then...Detection Method Uses Pros Cons Antigens HRP (P) Tyramide signal amplification of HRP Staining cultured...Enzymatic reactions on tissues can be cumbersome Tyramide reagents can be expensive Can only stain with ...of a nanobody that would make a useful RANbody, consider leaving a link to its sequence in the comments...immunodetection If you enjoy DIY lab projects, consider making you own DNA ladders with these plasmids...

... 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield imaging... which usually are undesired properties. In the ideal situation, a fluorescent proteis should emit a stable...since different fluorescence imaging strategies use widely varying amounts of excitation power (Shaner et ... intense light (for a brief time), whereas in widefield imaging fluorophores are excited with relatively...measurements and hope this information can help guide your understanding of reported data. Photobleaching...Measuring photostability at realistic powers will provide better insight into the photostability in the intended...situation. Measuring photostability in living cells provides a much more realistic view of photostability. ...

...missense variants identified from 58 individuals with GNB1 disorder since the identification of this disease...and Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is...other symptoms as discussed in this educational video. “SLC6A8 belongs to a large family of solute channels...perform a functional screen in mammalian cells to identify any solute channels that can compensate for the.... “This proof of concept study will help in identification of new targets that could greatly enhance our...keratinocytes,” says Mellett. This would help the lab identify therapeutic targets in the future. Congratulations...will express these candidate genes in TS iPSCs to identify whether any rescue cell migration and contraction...

...secondary considerations to validation, application specificity, and experimental considerations - no other...you know which ones to try?  Read on and we will provide you with some advice on the antibody selection ...antibody first look at the application-specific data provided on the manufacturer’s website. Because of how ...has been validated in your specific application. Consider also that there may be variations within an application...section then a directly-conjugated antibody will provide better resolution as these immune complexes are...abundance on a Western blot, a secondary antibody is ideal as it will help to amplify the signal. Some applications...the species that the antibody cross-reacts with. Ideally you will find an antibody that has been validated...

...enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides modulate...live cells. His work focuses on the enzyme hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase... susceptible to redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...she studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute ...neurons at specific firing frequencies. She hopes to identify how in vivo firing frequency of PV neurons affect...genetically encoded voltage indicators. Tian developed a video-based high-throughput screen for directed evolution...

...-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how gender...capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—...between male and female cells. The result of not considering the sex of the stem cells was that their male... in Norway, the team realized they should also consider the sex of the stem cells. In this case, they ... many researchers I’ve talked to who’ve never considered the sex of their cells. For one thing, cells ...hard to determine. To remedy this, Shah et al. provide a table indicating the sex of the most commonly... there are numerous methodological articles to consider—we have loaded those we are aware of in our checklist...

... Time project provides a framework for these undergraduate research questions. The idea is straightforward...were generating huge amounts of data that could provide the basis for student projects in introductory .... Genome projects use computational methods to identify genes by their similarities to genes in other ...studies generally leave questions about gene function wide open. What if two seemingly similar proteins have...phenotype is corrected, students can be relatively confident that the gene from the test species has a similar...Education: A Call to Action, Washington, DC. 2. President's Council of Advisors on Science and Technology...about Synthetic Biology with BioBuilder Use Addgene Videos to Learn about Basic Lab Techniques High School...

...trials involve identifying a therapeutic gene, finding a means to deliver it and identifying a suitable route...technologies have allowed scientists to quickly identify underlying genetic causes of some human conditions...example, it was possible to transduce the T-cells outside the body and thereby correct the mutations using...productive infection. AAV vectors are engineered to provide additional safety benefits: they lack all viral...immunity. AAV vectors are also capable of infecting a wide range of cell types including but not limited to...through direct evolution, capsid shuffling, and peptide displays. These capsids will be selected both for...to other gene delivery tools that are under consideration, recent advancements in various AAV technologies...

...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...

...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...

...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...

...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...

... 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 ...

...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 ...

...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 ...

...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.  ...

...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...

...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...

...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,...

... 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...

...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...

...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...