...Over the past decades, neuroscientists have made great strides to map brain regions and allocate specific...for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...surface. The latter reflects a state of behavioral despair, and the more time the animal spends in the passive...that were not (Hare et al., 2019). The research described here is only a small selection of the increasing...

...second outcome and increases the generation of the desired T-A base pair. Figure 1: Transition mutation...by base editors. Pathways that compete with the desired outcome are not pictured. Adenine base editors... SpCas9-VRER TadA-TadA* NGCG 8  Tips for designing base editing experiments in plants Choose a base...successful base editing causes the creation or destruction of any restriction enzyme (RE) recognition site... indel sometimes may also results in RE site destruction. Many thanks to our guest blogger Kutubuddin...

...Engineering Solutions, which also includes our CRISPR knockout guide design tool, guide validation tool, and...their relative abundances.   The ICE tool also provides a Knockout score (KO-score) which represents the...the CRISPR workflow. For a multiplex sample, ICE includes visual representations of all detected edit types...Contributions, Indel Distribution and Traces - provides particular details about the indel profile of ...

...1: CROPseq-multi uses two sgRNAs with internal barcodes (iBARs), multiplexed using tRNAs, within the lentiviral...separation of the multiplexed elements. The team designed sgRNAs that include an internal barcode and separated... have been made since prime editing was first described in Anzalone et al., 2019, including modifications... sparsely-labeled field of view that was more desirable than having many closely-packed labeled cells.... combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia research...

... defined as microbial survival despite exposure to antibiotics designed to kill them or to impede their...that the term ‘antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because...different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria that...equipment, etc. Virtually any preventive measure designed to help keep you from getting sick can be helpful...

...They also host AMA forums where users can ask designated scientists anything about a particular topic....calendar invite over to the r/Science team with a description of our AMA and Joanne’s preferred time to start...was writing all day and straight on through her designated two hours for answering questions. She was able...toxic advisors, fruitless job searches, and even despair at having chosen an academic past. I hope I was...receive via email. The cool features are too many to describe here, but I have over 2,000 notes which Evernote...

...data from your FRET experiments. You may have to design several different constructs to find one that could...studies. These parameters are less important when designing a biosensor, as the distance between the two fluorophores...gene of interest: Plasmid Color Expression Description pPROEX Aqua Cyan Bacterial Expresses Aquamarine... straightforward and quantitative, but it is destructive and cannot be used for dynamic measurements. .... Extra care should be taken so as not to destroy the donor molecule. Fluorescence lifetime imaging microscopy...

...insert into RNA. In practice, the term "promoter" describes the combination of the promoter (RNA polymerase...   Primarily used for RNA     transcript     Description Expression Additional considerations CMV General... phosphoglycerate kinase gene.  Constitutive  Widespread expression, but may vary by cell type. Tends ...promoters     Promoter     Primarily used for Description Expression Additional considerations T7 in ...identify the right tissue-specific promoters as described by researchers at the NIH here.  Note: A. Max...

...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012... bond between the VL and VH domains. This bond provides stability and solubility for the scFv (Gaciarz...clinic (Bates and Power., 2019).Many of the scFvs designed for oncology applications are reformatted as bispecific...Chantima W, Sangma C, Poomputsa K, Dharakul T (2015) Design and Generation of Humanized Single-chain Fv Derived...

...when it comes to fluorophore selection for panel design. Find streptavidin plasmids and biotin plasmids... this system is increased flexibility for panel design. Another perk is that one primary antibody can ...for example, a previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs...walk through the topics we covered in this post. Besides direct labeling, indirect labeling can be used ...surface, dump gating is a useful way to get rid of undesired cell populations. Dump gating uses the same color...

...protein analyses using ELISA. Peptides 72:4–15 . https://doi.org/10.1016/j.peptides.2015.04.012 Burnette WN ... Figure 2: The western blot dry apparatus includes a stack of filter paper, a copper anode/iblot ...blocking buffers, and incubation times. If the vendor includes publications that cited the antibody, consider...abundance In addition, the thickness of the band provides information about the relative abundance of the...

...identify possible gene function. In this post, I will describe a useful and cost-effective method that our lab...and probe with live cells The following is a description of a technique published by Simpson, Sacher, ...ends) and ligate into a bluntly digested vector designed for protein overexpression in E. coli, such as...the original plate to identify which phage gene encodes that protein! First, block your membranes with ... can lead to high background that obscures the desired signal. We have successfully corrected this by ...

... the work, shared the credit, and we both won accolades that would launch our careers in science. Fast...helped assemble a crack team of Addgenies that includes: Michelle, the Senior Scientist who coordinates... a happier person. What does a good team need? Besides that all-important sense of purpose, there are ...Slack constantly. Whether your teammates are two desks away or on the other side of the world, communication...self-interest ahead of the team’s goals, then that team is destined to do poorly. To some extent, it doesn’t even ...

...critical research infrastructure that has taken decades to establish.These funding decisions will have ...community for years to come. We Are Here to Help Despite these challenges, Addgene remains steadfast in ...educational resources: Our eBooks, videos, science guides, and protocols remain freely accessible to all ..., helping teams to be successful in their work despite limited resources. These resources become even ...

...right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself having...want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you have the...your membrane! Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane 8. ... with smaller tasks, or simply enjoy some well-deserved down time while waiting for your timer to ding...things you’ll want to consider (see our “technical design of a western blot”) post, most imaging issues can....2011.06.001 More resources on the Addgene blog Technical Design of a Western Blot   More resources on addgene.org...

... and the Puromycin resistance gene as separate peptides. PX459 This is the vector backbone. This particular...Protocols Paper for more information). V2.0 This designation separates pSpCas9(BB)-2A-Puro (PX459) V2.0 from... the Zhang Lab's Nature Protocols Publication describing them here. References 1.Ran, F. Ann, et al. ...Guide Pages Browse Our CRISPR Collection Find gRNA Design Tools ...

...select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting...protein, enabling the labeling of protein substrates/peptides and antibodies. Example applications of this include..., which is an important consideration for assay design (Breen et al., 2016). When selecting dyes, it's...fluorescent donors and acceptors in their product descriptions, or technical manuals. Specific applications...signal-to-noise ratios. Certain dyes have been specifically designed for use in super-resolution microscopy techniques...

... producing a wall of results that can only be described as bigly. But such a project should not be undertaken...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using CRISPR...References: 1. Doench, John G., et al. "Optimized sgRNA design to maximize activity and minimize off-target effects...with CRISPR/Cas9 Get John Doench's Tips on gRNA Design Check Out News Ways to Use CRISPR for Single Base...

...lab earlier, to read that stack of papers on your desk, and to tackle some new experiments before the next...gathering that pile of lab notebooks and binder on your desk. Arrange them in chronological order (or by project...labels. Color code your Post-its and write brief descriptions on each. Place the notes sticking up out of ...colleagues. Start small by identifying the protocols, guides, or references that your labmates ask to borrow...

...a job search website like Indeed. Do the job descriptions sound interesting? Is the work something you... types of jobs people with master's have Job descriptions often fail to capture all of the responsibilities...responsibilities of a role, so it helps to see how people describe their actual work. To do this, try looking at ...and industry, often means moving from the lab to a desk, although there’s often technical career tracks ...

... the parts of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas...DNA sequences. In laboratory settings, an sgRNA includes the crRNA and tracrRNA sequences in a “single-... sequences in the CRISPR array that will become guides for future targeted degradation. Before integration...biology of Cas9. Want a simpler guide to keep at your desk? We’ve got a CRISPR cheat sheet for that! Resources...

...know that the Fc domain provides structure, determines isotypes, and provides a place for secondary antibodies...yes, it does! While the Fc domain’s functions, besides structure and isotype determination, don’t generally... 2022). So the next time you see an antibody description with the Fc domain as only providing structure...

...database describing nearly 145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all ...authors of the paper in which the cell line was described. Using Cellosaurus Cellosaurus is searchable by...Cellosaurus received a report that HT-55, which was described as a colon cancer cell line, is actually a rectal...

...vast collection of empty backbones that have been designed, tested, and published by academic scientists....find the vector that fits your experiments, I've described below some of the most frequently requested vectors...protein with a specific resin (e.g. 6xHis containing pDEST-HisMBP). After purification the protein can be released...vectors. Consider your applications and experimental design. You can create a new strain with a disrupted gene...

...errors in the design of cloning strategies set experiments back days or even weeks. Primer design was done ... alleviate these problems through good software design. But what makes software good? Fortunately, that...we dug into the original literature, some of it decades old, to provide reliable feature annotations. Even...

... What are epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein...purification or detection of the tagged protein. These peptides consist of as little as 8 or as many as several...need monoclonal GFP antibodies or guidance on designing a GFP tag Addgene has you covered!   Potential...Protein folding issues, steric hindrances, and destabilization can be brought about by the tag so it’s important...

...resources to provide them to their students. My desire to create a set of low-cost, easy-to-use educational...protein – you first need the DNA sequence that encodes it. That DNA sequence then needs to be incorporated...one is as cheap as 10 cents, making activities designed with FD-CF reactions affordable to many schools...students. In our BioBits kit for example, we’ve designed FD-CF experiments that result in outputs that ...

...emission at 560nm. Shortly after the initial article describing the cloning of firefly luciferase was published...the plasmid, which is then introduced into the desired mammalian, plant or bacterial cells. Expression...stop codon, which is then transfected into the desired cells. A hybrid mRNA transcript containing luciferase...expression and more complex cell treatments can be designed to further explore the pathway. Which luciferase...

...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...

...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...use Twitter as a scientist. While this post is designed with beginners in mind, there’s some great reminders...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...

...target specific mRNA transcripts for degradation. Despite how easy these experiments are to physically set...to prevent the incorporation of non-specific nucleotides into the dsRNA leading to increased RNAi efficiency... Lab’s recent Nature Biotechnology paper, they describe TurboID and miniTurbo, which utilize non-toxic...switch to be expressed. In this case, the gene encodes the LacZ𝛼 protein. Once expressed, LacZ𝛼 complexes...

...but can be minimized with high powered research designs. Unanticipated discoveries Finally, there are good...rates, should be reported as a new discovery that deserves confirmation with new data and a new, preregistered...Guidelines; preregistration to increase clarity in study design and analysis; removing publication bias with Registered...Science, Dr. Mellor worked with citizen scientists to design and implement authentic scientific research and...

...New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in...Like other Cas13 enzymes, the Cas13d orthologs described in these papers can independently process their... high targeting efficiency, both of which are described below. Yan et al.: WYL-domain-containing accessory...produced very similar cleavage products even when guides were tiled across a target RNA, indicating that...

...trillion connections (synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it... this can cause challenges for the experimental design as these vectors can exhibit spurious expression...efficiency can have a major impact on experimental design and interpretation of results. Take extra care ...References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant permits efficient retrograde access...

...Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study....e. there won’t be positioning effects). When designing experiments where you want to express multiple...wondering, “How do I know which of my flies have the desired integration?” Not to worry! In flies, phenotypic...

... NEB's Resource describing Dam and Dcm Methylases Discovery of DpnI Review describing dam-/dcm- E. coli...mutagenesis. During this process, incorporation of a desired mutation into your plasmid of interest by PCR generates...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which...

...purification is now a reality thanks to OptoNBs. Recently designed by the Toettcher Lab, OptoNBs are photoswitchable... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...blue-light induced binding. The lab used this general design scheme to generate two more OptoNBs, one against...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...

...cleaved precisely as predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified ...advantage over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors...the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely be adaptable...worries, I've summarized the three strategies described in this post in the graphic below - including ...

... for over 10 years. Many successful scientists describe having a “posse” of mentors as one key to their...Ellen Daniell. In “Every Other Thursday,” Daniell describes a group of 7 women scientists who met every other...increases the diversity of input and perspectives Provides a good mechanism for accountability that makes...leadership and communication skills. The leader also provides materials such as pre-reads, videos or exercises...

...shown in the figure below. Their most successful design utilized Magnet photoswitchable proteins derived...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...NPPOC-caged trimethoprim (TMP) Cas9 fused to destabilizing domains (Cas9-DHFR) Gene editing, transcription...

... as the OG (as the kids say) affinity reagents deserved to be celebrated, there are many other amazing...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...°C and can refold correctly after denaturing.  Designed ankyrin repeat proteins, or DARPins, are genetically...a wide variety of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells. They...

...molecular biology experiments was in the job description, but this skill is not just about reacting to...puzzling through your broader research questions and designing future experiments. Being able to work through...research non-academic careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined...most likely cause, you implement an experiment designed to fix the problem. Then you assess whether your...

...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...   Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral Tet-on ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

...another. If you are using barcodes (and you should be!) bear in mind that your barcodes are being applied to...containers, not to the contents. Tightly coupling barcodes and containers might not be such a bad idea, tightly...Resources on the Addgene Blog Learn about How We Use Barcodes in the Lab Get Tips on How to Keep a Lab Notebook...

... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool... efficiently, as those that do not contain the desired insert should not grow.   Figure 2: Image...

...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...Strong protein-protein interactions with other peptides. Created with BioRender.com.     Many biologists... conjugation. But hopefully, this introduction provides a good base from which to dive deeper!References...

... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...Addgene partnership to provide AAV viral preps, includes listing of transfer status for viral preps....vectors See Addgene's Current AAV inventory , which includes all viral vectors transferred from the Penn Vector...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...