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...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. ...

... 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...

... 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...

...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 ...

...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...

...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...

...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...

... 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-...

...are subject to excision and ssDNA cleavage by canonical base excision repair enzymes. At the same time...2019). TREE is a real-time, fluorescent based identification system for the isolation of base-edited cell...targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (BFPH66) contains a histidine...GFP positive cells isolated with TREE have a significantly higher frequency of genomic base pair edits ...GFP positive cells can be sorted and have a significantly higher frequency of base edits of interest than...robust induction of GFP fluorescence confirming the efficacy of GO as a base-editing reporter. As the initiation...markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter toolbox. ...

...: 18px;font-family: helvetica;","color":"#FFFFFF","variant":null,"font":"helvetica","style":"font-family...button_font":{"size":18,"styles":{"font-family":"helvetica","font-style":"normal","font-weight":"normal",...font-family: helvetica; font-style: normal; font-weight: normal; text-decoration: none","font_set":"DEFAULT"}... into viral vectors that can be administered systemically (retro-orbitally, RO; or intracerebroventricularly...intracerebroventricularly, ICV) or locally (stereotaxically, STX) to the brain. Figure 2: Selection, prep, and testing...developed, funded, and shared has no doubt changed drastically over the past few decades. But no matter how ...and transgenic mouse lines for genetic access to cortical cell types. Cell. https://doi.org/10.1016/j.cell...

...cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large in size...size and may have spread while Stage 4 indicates that the cancer has spread from where it originated to ...asbestiform minerals such as erionite, are the only scientifically proven causes of the disease. There is no question... question on this point within the medical and scientific communities, and the link between asbestos and.... New techniques are currently being tested in clinical trials to detect mesothelioma earlier than ever...earlier, thus improving the patient’s prognosis significantly, but also to monitor those who may be at greater... working to fight the cancer.  Beyond early modifications to standard treatments and early detection, ...

...Addgene ID number, your publication will be listed on our website as a “Publication Citing this Plasmid” ...upon other researchers being able to find their publications in the first place. As a curator of the thousands...make it easier for you to find the plasmids and publications relevant to your research interests. We do this... this by promoting citation, supporting sophisticated search, and curating plasmid collections. Addgene's...number as well as the reference for its original publication. To help facilitate this process, we explain ...fruits of your labor and won’t have to repeat the modification. This will save both time and money. A quick...whether the plasmid has been used in your system. Sophisticated search You can search for specific plasmids ...

...If you use a kit for DNA purification or if you use a DIY purification protocol, you might have noticed... mutations in single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade...degradation during plasmid DNA extraction and purification from E. coli is the presence of the protein ...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been...chelating agents that can be introduced in the purification protocol to remove endonucleases activity. For...directly use your DNA preparation in any downstream application involving enzymatic reactions, a valid alternative...temperature for a short period, and there are indications that Tris buffer is better than water in these...

...any DNA editing functionality Collecting preclinical and clinical data about a disease or therapeutic Side-by-side... ReFigure. Reading and exploration including replications and experiments resulting in “negative data”...small selection of data found in full-length publications. As early career researchers, we wondered how...performance as in this antibody ReFigure Analyzing replications as in this ReFigure about whether Natronobacterium...reagent performs well, or you might publish a replication experiment that you conducted as part of setting...papers as they are not included in traditional publications. By submitting your data to a repository (Figshare...-registration, preprints, data, software, micropublications) in grant proposals. ReFigures are one such...

...The biomedical field is often concerned with understanding the cause of diseases and how to treat those...optimization is performed to take the drug to the pre-clinical and human trial phases.   So how does CRISPR factor...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate...enables researchers to select and generate physiologically relevant models. In complex diseases, it can...patient mutations which are variants of unknown significance – mutations with unclear effects on disease ...standard is to test the drug on models which are genetically proficient and deficient for the target. CRISPR...

...gene at once. Therefore, applications that require simultaneous modification of all alleles of a gene,...frequency three orders of magnitude higher than identical targeting vectors delivered through transfection... recommends designing homology arms with the modification of interest, ordering them from a gene synthesis...with a homologous repair template. It is this application - the creation of sequence-specific knockins ...clones, making it unsuitable for most in vivo applications.  Third, even in the most optimized tissue-culture...tissue culture based approaches that require modification of only a single allele of a gene, such as the...efficiently deliver large repair templates for applications that require large gene fusions. The current...