...provided here for reference: OpenWetWare Preparing chemically competent cells (protocol)  Preparing electrocompetent...protocol)  Untergasser Lab Preparation of chemically competent cells (protocol) Preparation of electrocompetent...until we are ready to perform transformations. A typical 200mL bacterial culture produces 20mL of competent...transformation protocols below: OpenWetWare Transforming chemically competent cells (protocol)  Electroporation (...protocol)  Untergasser Lab Transformation of chemically competent cells (protocol)  Transformation of...plasmid is introduced to bacteria, the bacteria is typically grown in liquid culture without antibiotics to...

... why you might want to be careful relying on so called “housekeeping proteins.” Fully quantifying a western... later steps in this chain (blue) to infer or calculate things about earlier ones. In practice, multiple...popular choices for loading controls. These are often called “housekeeping proteins” and are detected with an... will almost certainly be overloaded. One paper called this problem “the most common error associated ...Data is presented as mean ± SEM. Adapted from Analytical Biochemistry, vol 440, Gilda, J. E., & Gomes,...loading control to β-actin for Western blots. Analytical Biochemistry, 440(2), 186–188. https://doi.org...Antibodies 101: The Basics of Western Blotting Technical Design of a Western Blot Troubleshooting and Optimizing...

...populations; for example, CD8+ T (“T killer”) cells. Typically, you would label the CD8 co-receptor protein on...of interest via FACS using negative selection is called dump gating. Let’s say we want to sort out all ...cell membrane but inside the cell. Due to their chemical nature, antibodies cannot penetrate the cell membrane...intracellular labeling with antibodies requires chemically pre-treating the cells to allow for entry of ...esterified dyes that can enter cells freely are typically used. Inside the cell, the ester group is cleaved...intracellular labeling protocol is necessary, requiring chemical treatment of the cells after surface labeling....Associate in the lab of Arlene Sharpe at Harvard Medical School. His projects include investigating negative...

...colleges, private universities, and medical schools. I will emphatically deny that I have a magic formula..." manual on applying for faculty positions in biomedical science. My motivation was to share my experiences...told all of the nuances of the research in as technical of language as possible. Seriously. My suggested... trying to say X." She would then look at me quizzically and ask why I didn't just say that? Bottom line...especially helpful. I set out to make something practical and accessible. If there's something you come ...the Janelia Research Campus of the Howard Hughes Medical Institute. His interests include the quality control...

...able to bind to a small molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone...intracellular locations using Cas9 and track the localization of the complex. Broccoli, the other green fluorescent...Broccoli, and useful for quantitative studies. Specifically, Corn was developed to quantify RNA transcription...high-affinity complex for RNA labeling and tracking." ACS chemical biology 9.10 (2014): 2412-2420. PubMed PMID: 25101481...and directed evolution." Journal of the American Chemical Society 136.46 (2014): 16299-16308. PubMed PMID...di-GMP and cyclic AMP-GMP." Journal of the American Chemical Society 135.13 (2013): 4906-4909. PubMed PMID:...a photostable RNA–fluorophore complex." Nature chemical biology 13.11 (2017): 1187. PubMed PMID: 28945233...

...where you can start to identify which items aren’t critical to your research success. If you can get away ...resources to keep, and which ones to abandon. It’s critical to make these decisions in the following order... budget can help you compare the monetary and empirical value that helps you determine whether or not ...know how to decide which components are the most critical to your research, negotiations can be much easier...negotiating finances, instead of solely discussing numerical values, try to keep conversations focused on research...saving the institute time and energy that would typically be wasted going through some of the questions ...is your goal, then understanding your needs is critical to a successful negotiation and collaboration....

...you’ve saved. Repurposing regulated medical waste Regulated medical waste (RMV), which we collect in those...scores products by their environmental impact on a scale of 1-10: the lower the score, the lower the environmental...companies to obtain these labels! Reusing plastic Historically, scientists washed and reused materials such...flow cytometers. These fluidic machines handle biological samples and are cleaned between each use. You...those red biohazard bags, typically is destined for a landfill. However, Triumvirate Environmental developed...

...deaminase (TadA) to a catalytically impaired Streptococcus pyogenes Cas9. They called this editor Target-...-to-inosine (A-to-I) RNA editor called REPAIR by fusing catalytically inactivated RNA-targeting Cas13 ...plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools more ...editor still retains A-to-I editing. This system is called RNA Editing for Specific C-to-U Exchange, or RESCUE...

...temporal and spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control...light-inducible heterodimerizing cryptochrome 2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins....this approach. This study generated the first optically active saCas9 protein. Stop light! Photoactive...different approach. These researchers used a genetically encoded photocaging technique to insert a light-removable...light-removable protecting group, specifically a nitrobenzyl photocaged lysine (PCK), on the Cas9 protein...Hemphill et al. presented data showing that this genetically encoded, photocaged Cas9 system could silence...editing and transcription. Label genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged...

...Regulatory DNA function is typically inferred based on correlative biochemical characteristics and ectopic...Canver of Boston Children’s Hospital and Harvard Medical School. Addgene is proud to present a video reprint...for deletion screening by conventional PCR. Biallelically disrupted cells can be easily identified. Even...investigator at Boston Children’s Hospital and Harvard Medical School. His lab studies the molecular genetics ...Matthew Canver is an MD/PhD student at Harvard Medical School in the laboratories of Stuart Orkin and ...

...Kristin Krukenberg and Jessica Polka from Harvard Medical School and a long list of supporters from all of...peer review and grant awards systems need to be radically overhauled.  Finally, the science publishing system...excellence to begin effecting change.  Panelists call for action Each of the speakers in the panel had...Director of the Office of Postdoctoral Affairs at UNC, called her presentation “Disequilibrium, Disillusion and...Associate Professor of Systems Biology at Harvard Medical School, titled her presentation “How to Train Your...and take action. More Reading: “Rescuing US biomedical research from its systemic flaws” by Bruce Alberts...

... then study where the protein is localized, and how its localization and expression may change across ...transcriptional activation. Multiple nuclear localization signals (NLS) increase protein import into the...technology, the Vale and Weissman labs took this biological lesson and created a system to amplify fluorescent...modified the GCN4 sequence to increase its alpha-helical structure and stability, creating v4 SunTag. Since... also affect protein activity, half-life, or localization, these concerns are greater with SunTag. v1 ...

...poster tubes (or as our baffled flight attendant called them, "long, skinny things") and generally exuding... tales of back country skiing, fly fishing, and local grizzly bear attacks. He described one such recent...All Addgene depositors! Addgene was mentioned specifically in Dr. Zhang's introduction. His willingness...Cas9 that is improving the nuclease specificity dramatically and reducing off-target effects. Other young...discussed some of the many challenges of creating biological circuits, and I can only say this science is ... deposited and distributed – have changed so dramatically in the past 4 years. I kept busy for the whole...

...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal...promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity to... the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...deadCas activator/repressor experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets...effectiveness as a control. Since off-targets typically confound an experiment due to their location (...

...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature...Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...

...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...varying ratios of recipient plasmid to insert. It is critical to set up a negative control ligation reaction...electro-competent cells instead of the more common chemically-competent cells. You should perform, at minimum...purifications, you generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic...

...an antibody that works for your application, specifically detects your protein, is species compatible,...along with some pros and cons. FLAG FLAG is a synthetically derived 8 amino acid tag (DYKDDDDK) that can...possibly interfere with protein function and localization. If you need monoclonal GFP antibodies or guidance... N or C terminus of a protein may disrupt the biological function of some proteins. Protein folding issues... In rare cases, tags can also affect protein localization and solubility, so be on the lookout for aggregation...Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol...

...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...science research all the way up to the production of clinical viral therapies. Protein production  HEKs are...recombinant proteins at high levels. In a case where clinical applications of recombinant proteins are necessary...also grow in suspension, which allows for large scalability, and can also be adapted for serum-free growth..., which can make for a seamless transition of biological tools to other researchers using the cell line...

...arguing with me. First I calmed the situation down by opening a dialogue specifically about the project and...We spoke about it for a while and he eventually calmed down, apologised and admitted he’d been under a...wherever it makes sense to start and try to be as chronological as possible — if you find yourself jumping backwards.... Your story should be too. Use drama Use any rhetorical technique you have in your arsenal to make sure...start point and it needs a good end point. One thematically pleasing option is to end at the present — ‘...

...Extremophiles - phylogenetically diverse microbes living in extreme physical and chemical conditions like...Stanford University became interested in exploring biological dark matter – large tracts of the microbial tree... to be diversity hotspots containing abundant biological dark matter and so the lab organized a sampling...like YNP thermal sites - are also of practical interest since they’ve previously yielded valuable enzymes...

...algorithms are trained on vast amounts of data to calculate the relative immunogenicity score (Mattei et.al... evaluation of ‌therapeutic immunogenicity is typically performed using traditional 2D assays with immune...inform decisions about in vivo application and clinical testing. This would pave the way for improved ...in Macaca fascicularis: From Manufacturing to Preclinical Studies. In J. Bluemel, S. Korte, E. Schenck,...F. Weinbauer (Eds.), The Nonhuman Primate in Nonclinical Drug Development and Safety Assessment (pp. 519...2016). Antidrug Antibody Formation in Oncology: Clinical Relevance and Challenges. The oncologist, 21(10...

... transcription on the array have limited the scalability and efficiency of multiplex gene editing. Moreover... fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. They...been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the tandemly assembled...released by endogenous tRNA processing machineries, specifically, RNaseZ and RNaseP. Although the Gao Lab demonstrated...loci MPE with similar high efficiencies, which is scalable for a higher number of MPE. Therapeutic potential...drive and process the tRNA-gRNA arrays, which is scalable and efficient in plasmid and viral vectors. In...

... from their original sources. Figure 1: Biological repositories that distribute mouse strains, plasmids... plasmids, cell lines, and much more have been critical for pandemic research. Image from Reproducibility...Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition in Japan. These... available to scientists around the world. This scale of production and distribution is possible due to...research, and mechanisms to maintain and grow these critical organizations should be a fundamental part of ... SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory...

... Social distancing? Many people are now calling this “physical distancing” since it’s important to stay...resources collated by Addgenies to help you keep calm and science on. Practice self-care and stay connected...in these is a great way to support your favorite local exercise spot or find something new. We usually ...message for some people. We should recommend #PhysicalDistancing. Social interactions and #social support ... COVID-19. You can also give back by supporting local businesses (ex: purchasing a gift card to use later...

...Musicophilia includes case studies of relevant neurological atypicalities, including a patient with amusia - the...capture the attention of even the most skeptical reader of historical fiction (I can personally attest to...Science is a terrifying and equally good wake up call about the science of race."     - Joanne Kamens,...Strange & Mr. Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the...

...many opportunities available to those who are scientifically minded! Listen to Biotech Founder Michael Koeris...required Research skills The ability to think critically and carry out in-depth research is applicable...Scientific Advisor Lab Manager Patent Lawyer Technical Writer Meteorologist Journal Editor Textile ...organised, reliable, and can work independently. Practical outdoor skills are needed in a variety of roles...Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist Documentary Director ...range of roles, including: Science Reporter Technical Writer Statistician Bioinformatician Science...

...conditions/locations but to engage the local community (including local garden and compost clubs) to participate...easy. Most of our equipment has been donated by local companies and BosLab has grown into an organization... for discovering novel therapeutics. Dave is a chemical biologist with a Ph.D. from UNC-Chapel Hill and...but are inspired to get involved, check out the local groups page on DIYBio.org to find a community lab...and follow us on twitter or facebook. If you are local, join us for our next event on meetup.com! Many...

...targeted cell ablation via ROS is achieved through localized expression of the FAP. A new photosensitizer:...the main advantages of MG-2I-dL5** over other genetically encoded photosensitizers is that it works with... ROS because NIR light doesn’t excite natural biological chromophores found in cells.  He et al also show...compared MG-2I-dL5** head-to-head with KillerRed, a genetically encoded red fluorescent photosensitizer protein...Watkins, S. C., . . . Bruchez, M. P. (2016). A genetically targetable near-infrared photosensitizer. Nature...Chepurnykh, T. V., . . . Lukyanov, K. A. (2005). A genetically encoded photosensitizer. Nature Biotechnology...

... Connie Cepko (Harvard Medical School), and Dr. George Church (Harvard Medical School) for taking the ...environment? Does the group expect each other to be physically present at certain times during the day? We asked...history in science. Potential graduate students typically go through a rotation, which reveals their working...job satisfaction (especially for scientists and technical people) is linked to engagement and access to ... "chaotic" and offer less micromanaging, they typically offer more flexibility in choosing direction of...

...such as plasmids) into mammalian cells, a process called transfection. Transfection is somewhat comparable... way. Selection methods, described below, are typically employed when creating stable cell lines and are...respectively). Therefore, it is not always mission critical to make sure the plasmid replicates in the cell...   ***The concentration used for selection is typically more (double) than that used for maintenance of...usually achieved by performing a "kill curve" (basically growing cells in various concentrations of the...resistance; however, like gentamicin, neomycin is typically used to suppress bacterial growth. The selection...

...specific subcellular localization peptide. By combining three different localization peptides with four ... for example).  Given that the liver is an immunologically active organ, this advance is likely to afford...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...system. dCas9-SSAP will make the editing of kilobase-scaled sequences more efficient and less risky.   ...plex mixed-subclone system using mStrawberry, localization peptides, and reporters that drive the expression...

...variables - your approach, your workflow, or your physical setup - to prevent the mistake, just like you ...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists...light-sensitive reagents” or “re-sterilize now.” Have a physical copy of the protocol when you work and make a ...time to do them carefully and thoroughly. Have a physical copy on hand as you run your experiments. Try ...your plate maps onto your plates, alongside your physical copy. If you’ve got messy handwriting, reach for...accuracy.  Bench setup Small adjustments in the physical organization of your workspace and your workflow...

...This process is called α-complementation. The system described above was put to practical use in the following...well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme β-galactosidase...β-galactosidase enzyme can break down a dye-linked substrate called x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside...deleting a section from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase...Providing DNA encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell...

...proteins yet they still share many of the same technical advantages that come with working with prokaryotes...mutation. Yeast expression plasmids used in the lab typically contain all the necessary components to allow ...are small independent chromosomes and are thus typically found as a single copy. Unlike the ARS vectors...promote replication.  Selection markers for yeast Historically, scientists have utilized auxotrophic selection...plasmid may be expressed at higher than normal physiological levels due to high copy numbers. This creates...the presence of the selection marker at non-physiological levels. Scientists have tried varied approaches...

...manipulating interneurons using a viral approach Genetically modified organisms like transgenic mice are important...a synthetic red fluorescent protein variant they call mScarlet. Bindels et al used a combination of deliberate... cell physiology and molecular processes via genetically encoded proteins. PhoCl is a new member of this... such as an NLS or NES, enables control over localization. Inserting PhoCl between an enzyme and an inhibitor...experiment, check out our recent guest blog series: A Practical Approach to Choosing the B(right)est Fluorescent...investigated an aspect of telomere length regulation called telomere trimming and have proposed a role for ..., Li and colleagues observed endogenous TZAP localization at telomeres and found that TZAP binds directly...

...composed of four peptide chains (two identical heavy chains and two identical light chains) each with a constant...antibodies means that it is relatively easy to physically divide up the different regions, both through...joined by a linker peptide, resulting in a product called a single-chain Fragment Variable (scFv). Size: ...samples more efficiently and can also improve localization accuracy when used in super-resolution microscopy...David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8(...

... audience and convey your ideas clearly and strategically is powerful in almost any career. It allows ...my head. What came out, though, was an incredibly calm: “Excuse me, I seem to have lost my place. I need... the open windows in the middle of your talk. Technical difficulties turn your slide deck into a chalk...everyone! Everybody makes mistakes, encounters technical difficulties, and runs into unexpected stumbling..., to a group of peers who are there to provide critical feedback, often with a semi-international audience...your own native language. And, at least in the biomedical field, with very little text on the slides and...

...limitation and allows for the flexible design and small scale production of antibodies. Problems with antibodies...instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated to reporters...labeled tyramide molecule into a highly reactive radical, which is then covalently linked to residues within...however, compatible with most multi-labeling immunohistochemical protocols where either a directly labeled... required for detection RANbody is detected enzymatically with HRP substrate More sensitive than detecting...RANbodies) as versatile, small, sensitive immunohistochemical reagents. PNAS. PubMed PMID: 29440485. Additional...

...Mother Nature’s toolbox. Natural plasmids, also called environmental plasmids, are all around us! A plasmid...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cells survive their own secretions. Pro tip! Historically, colicin-producing plasmids have been referred...which strains of E. coli contain this F plasmid is critical for successful Gateway cloning. In addition, scientists... Vogel, T. M., & Simonet, P. (2001). Laboratory-scale evidence for lightning-mediated gene transfer in...Characteristics and Potential Applications in Biotechnology. Critical Reviews in Biotechnology, 24(4), 155–208. https...

... be used for measuring protein expression or localization in cells, or even single-particle tracking of...named according to the number of strands in the canonical beta-barrel structure. Most frequently, the proteins...fluorescence complementation analysis under physiological conditions. Shyu YJ, Liu H, Deng X, Hu CD. Biotechniques...fluorescent proteins as tags and biosensors for multiscale imaging. Shcherbakova et al. Nat Commun. 2016...split-FP options. While the BiFC interaction is typically irreversible and requires some time for chromophore...