...propagated it, separated it into roughly identical replicate groups, and treated these groups with the...among the separate replicates would have different labels - i.e. they would stochastically arise from different...summarized in a recent Nature Medicine paper. In the publication, Bhang and coworkers used the ClonTracer library...below): 1. Resistant cells among the separate replicates would contain the same labels - i.e. they would... Looking at barcode abundances among multiple replicate populations, they found that similar barcodes ...populations were shared by at least one other replicate; prior to treatment, there are small (previously...such that initial resistant populations can be eradicated up-front quickly. This approach is in contrast...

...such as affinity purification mass spectrometry (AP-MS), lack the ability to specifically identify proteins...the validation of a binary PPI as well as the identification of additional interacting factors. Problems...detect weak and/or transient PPIs. Many PPI identification techniques rely on protein interactors to stay... AP-MS. This requirement often prevents the identification of weak and/or transient interacting proteins...lysed before the bait protein is pulled down, typically with an antibody. This can artificially select...controllable version of the BioID protocol. The critical component for both BioID and Split-BioID is BirA...Further characterization of these candidates is critical to understanding how they interact with the bait...

...past decade, primary cells remain difficult to genetically edit due to toxicity from electroporation or ...some cases) and minimal toxicity, including in clinically-relevant lines such as CAR-T cells. Plus, this..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...library generation and screening that led to identification and characterization of AAV.CAP-Mac. Image ...endothelial cell-specific tropism, with slightly reduced efficacy compared to AA9-X1.1. Importantly, AAV1-X1 is ...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...

... utilize an array of tools that rely on DNA amplification and manipulation to produce and edit plasmids...plasmids. These are prone to error — both human and biological. Even the most high-fidelity polymerases can ...Potential points where DNA errors can be introduced, indicated by exclamation marks. Error points include places...places where DNA is being created, such as PCR amplification or bacterial growth. Created with BioRender....the correct inserts, tags, promoters, and other critical functional components. Fun fact! Some older plasmids...sequences. These sequences can also be confirmed or theoretical. No matter the level of information that is provided...elements, and nucleotide indels or mismatches in significant plasmid elements. Which differences are considered...

... series.  “The only healthy communication style is assertive communication.” - Jim Rohn, Author, Entrepreneur...Assertiveness? An assertive person is able to communicate their ideas confidently while also considering...of the most frequent sources of conflict is miscommunication. For example, you might misunderstand your...the wrong direction. The best way to avoid miscommunication is to follow up after every meeting with an...workplace, these strategies will help you to communicate with confidence earn respect from your supervisor...in an email, there is a high likelihood of miscommunication, which can lead to even more conflicts. If...manager, you can resolve most conflicts if you communicate your ideas assertively, listen to his or her ...

...lines of communication Few people actually like meetings, but run well, meetings can be a critical part of...depositors; Gizela, the lab member who knows how the physical kits are made and shipped; Chiara, the tech transfer...transfer expert who understands the somewhat complicated Material Transfer Agreement process that kits...repository, performing quality control, and providing technical customer service. Team members are highly trained...But this is a blog post, so here are just a few critical features based on my own experience: Leadership...should be on the team. As a team leader, I’ve (diplomatically) booted people off teams. I’ve also kicked ...are helpful, they are certainly not the only communication options. Addgene, along with an increasing number...

...the cell type under study. Many AAVs will show significant expression in a variety of cell types, but you...previous literature using AAVs for DREADD delivery indicates ~7-21 days are required for the onset of gene ...rate limiting step and can immediately undergo replication and transcription. However, with scAAV the effective...by the use of AAVs? 1) Combined functional and anatomical mapping of circuits In our lab, we use AAV-DREADD...these complexes are so robustly expressed that anatomical assessments can be easily performed too. While...identified this feature of AAVs as a new tool for neuroanatomical tracing (11). With the ability to produce neuron...Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered receptors (i.e. chemogenetics vs. ...

... difficult to eradicate. You can suppress the growth of mycoplasma but cannot eradicate it completely....Contamination! Tissue culture media often have indicators included in the media that turns yellow from ...contamination Detection of mycoplasma by naked eyes or optical microscope can be very difficult, so how do you...cells detect mycoplasmas, they trigger a series of chemical reactions and this induces a noticeable color ...contamination, before using the cell lines for assay or analytical purposes. Use 0.1-micron filters as are they ...Fumigate the laminar airflow and the lab facility periodically. The CO2 incubators, used for maintaining cell...cleaned regularly and checked for contamination periodically. For more tips, check out Addgene's video Getting...

...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they... it integrates, which produces a target site duplication (TSD). Created in BioRender.com.   Autonomous...transposition activity  Tol2 transposon plasmids    Applications of transposons Now that you’ve learned about...

...Sontheimer EJ, Mello CC (2018) 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome...repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x and 3xFLAG epitopes...Mello lab also reported that modifying the 5’ modification of oligos used to generate linear dsDNA repair...melting and rapidly cooling dsDNA repair templates dramatically boosted editing efficiency (Ghanta et al., 2020... more efficient. Pure PCR repair templates are critical for efficient knock-ins (Ghanta et al., 2020)....Farboud et al., 2019). Work from the Mello lab has dramatically improved RNP editing with linear repair templates...Efficient Marker-Free Recovery of Custom Genetic Modifications with CRISPR/Cas9 in Caenorhabditis elegans. ...

...one another, and a value of “1” indicating those markers are identical and completely indistinguishable... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...relies on “unmixing.” Unmixing uses a complex mathematical algorithm and single stained reference controls...spectral flow experiment and panel, there are a few critical factors to keep in mind in order to get the best...autofluorescence across various tissue types varies, it is critical to include an unstained sample for each tissue...complexity index ranges from 0-1, with a value of “0” indicating those markers are completely distinct from one...samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies ...

... molecular biology-grade agarose containing physiological concentrations of salts and glucose. If we circled...diluted from 10X and containing both Hepes and bicarbonate) is brought to room temperature and placed in..., and making sure we always maintain a backup replicates of each clone in case of contamination. We do...fluorescent protein), fluorescence microscopy can theoretically be used to select specific wells from the 48...further analysis. To assess CRISPR/Cas9 knockout efficacy, we have used both Western blotting and PCR, depending...population of transfected clones. The technique is applicable to a wide variety of cell lines, from HEK to ... terms of growth rates, morphology or other physiological properties. An advantage of this technique is...

...in normal biological processes and disease states. While LITE plasmids have clear application to anyone...effectors (TALEs) as anchors for genome engineering applications was still very new. Now, Konermann and her colleagues.... All of this requires genes to be regulated dynamically.” To understand how that works in memory formation...they could package LITEs in viral vectors and genetically target them to specific cell populations. They... the brains of living mice both quickly and significantly. Genes under LITE control showed a rise in transcription...prove the causal role of specific epigenetic modifications in the genome,” Zhang said in MIT’s press release...with LITE? Reference: Silvana Konermann et al. "Optical control of mammalian endogenous transcription and...

...? The available filtering options now change dynamically based on the type of material and specific search...articles. From paper to plasmid Authors regularly indicate that their plasmids are available from Addgene...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...guide RNA / shRNA inserts Resistance Marker: This indicates the selectable marker used in your model system...Requests: Only see plasmids with more than the indicated number of requests Availability to Industry: Restrict...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...(Prussin et al., 2015), only a small fraction typically cause disease. The gamma-retroviruses have their...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...of a non-living viral particle, and how we as dedicated researchers can exploit it. This blog was written...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral... proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375–394. https://doi.org/10.1128...

...MCC develops and makes freely available technical and pedagogical resources, offers mentoring for instructors...best teachers and mentors pass on more than just technical knowledge, shaping their trainees’ very way of...citrate synthase and presented their findings at the American Society for Biochemistry and Molecular Biology...their research capabilities and laboratory and critical thinking skills (Knutson et al., 2010a,b). Classroom-based...implementation. Funded by the NSF (with Co-PIs Jessica Bell, Lisa Gentile, Ellis Bell, and Amy Springer...Einstein: A Life. Chapter 17, Einstein Discovers America. New York./  Callahan, K., Peterson, C., et al....biochemistry laboratory: Part I – guided inquiry – purification and characterization of a fusion protein: Histidine...

...candidate in the Sternberg Lab at Columbia University Medical Center. DNA transposons are ubiquitous genetic ... and have been adapted for a wide variety of applications - as mutagenesis tools for genetic studies (... The process also creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family...a multi-spacer CRISPR array. Workflow and technical considerations Making insertions in bacterial ...into cells and plate overnight Screen colonies phenotypically or by PCR/sequencing While CRISPR-transposons...few important details. For transposons to be specifically recognized during transposition, genetic cargos...Leveraging a unique marriage between distinct biological elements, CRISPR-transposons excel at programmable...

... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...expression and correct folding, post-translational modification, protein degradation, access to the nucleus ...organisms, including bacteria, alternative fungi (C. albicans, pC2HB) (7) and mammalian cells. If the problem...co-expression of the enzyme responsible for the modification in the assay host strain can help. In order...that the configuration of your fusion proteins physically blocks the binding sites for the protein partner...Tachihara, K., Umezawa, Y.l. Split luciferase as an optical probe for detecting protein-protein interactions...

... at Sanofi Pharmaceuticals. This post highlights a rare disease, Fibrodysplasia ossificans progressiva...surpassed 60,000 views for the month of September! Historically we do better in September than in the summer...or you can’t quite remember the hazards of that chemical in your obscure growth medium, these apps can ...Binding Proteins," was contributed by guest blogger Jessica Sacher from the University of Alberta and describes...the post for the full details and many thanks to Jessica for all of her hard work and attention to detail...can find the rest of our September posts in chronological order here: Four Things I Learned at the 10... a Lab and a Project That’s it for now. As I indicated in the opening paragraph, I’m currently on my ...

...were less significant than when non-targeting guides are used as controls. Less significant p-values lead... approach for identifying genes involved in a biological process. These screens provide an alternative...highly effective, are affected by low on-target efficacy, non-specific toxicity, and off-target effects...calling a gene a hit when really it’s not). In practical terms, this means that using safe-targeting guides...guides and may more accurately determine the significance of hits in non-growth screens. Off-target cutting...of on-target activity. However, there was no significant difference in depletion of ricin sensitizer genes...and do not have a major reduction in on-target efficacy. Results from Morgens et al. show that genome-...