...knockout, activation, repression, and barcoding.  3) Are you trying to target every gene in the genome...sequencing. Representation - Most libraries contain 3-6 gRNAs per target gene, and maintaining the distribution...preparation from Addgene, you can skip the steps above! Cells are then transduced with lentivirus containing...practices, CRISPR libraries can help you learn a lot about your phenotype of interest. For more detailed information...Libraries” (McDade et al., 2016). To learn a bit about how CRISPR/Cas9 can be used in other types of experiments...

...plasmids. Second generation packaging systems use 3 plasmids; an envelope plasmid that usually encodes...vessel size. Opting for a larger vessel, such as a 3 or 5-layer flask will require some initial optimization...available systems. Producing lentivirus: it's all about the T's (transfection and titers) To produce lentivirus... optimize transfection conditions. As mentioned above, plasmid ratios can be adjusted to enhance titer...

...biological study without her or her family's knowledge. 3. Fossil Rock Anthem One of Tom's earliest and most...Science with Tom. In this podcast, we learn more about Tom and pick his brain for advice on how to dive... Rap of Henrietta Lacks Students in Oakland rap about Henrietta Lacks - the African American woman whose...Please Don't Kill My Hive Beautifully filmed video about colony collapse disorder.     5. Plasmid Rap ...science standards and communicate science. Learn more about Tom on his youtube channel and at sciencewithtom.com...

...passion and pay attention to the details. Chapter 3 - Answering interesting biological questions using...of our educational resources. Listen to hear all about Professor Goedhart, his lab, and some of the tools...

...degradation of endogenous proteins (notably, BRD2/3/4, CDK9, TRIM24, FLT3, BTK, and ALK) by linking small...with dTAG-13 at doses as high as 20 µM (see Figures 3 and 5 in Nabet et al., Nature Chemical Biology for...degrade any protein. We are particularly excited about the dTAG system as we can use it to evaluate the...

...targeted sequence at the locus of interest. SpCas9’s 3’ NGG PAM occurs frequently in GC-rich genomes, but...100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent editing...Blog Learn about Engineered Cas9 PAM Variants Read about CRISPR Base Editing Learn about Homology Directed...editing specificity. We’re excited to learn more about xCas9 - here’s what we know so far! Why do we ...point mutations, also has a strict editing window about 15 nt upstream of the PAM. Even if there’s a PAM...in mammalian cells, finding xCas9 3.7 displayed about the same editing rate as SpCas9 at an NGG PAM. When...are exciting, there’s a lot we still don’t know about xCas9. “I’m not 100% sure xCas9 is going to be flat...

... of the optical fiber into the region of interest 3) Behavioral experimentation I’ll discuss some of the...thoroughly elsewhere (here and here for instance) [2], [3]. Karl Deisseroth of Stanford University , one of ...10.1146/annurev-neuro-061010-113817. PMID: 21692661.  3. Bernstein JG, Boyden ES. Optogenetic tools for analyzing... electrophysiology data too but that’s what collaborators are for). For example, if you study neural pathways...intended brain region and/or cell type (I’ll talk more about this in detail later). But what are the different...of your opsin, but keep you eyes open for posts about the next steps in the coming weeks. Expression of...Addgene. See the Deisseroth lab homepage for details about types of constructs available, instructions for ...

... The collaborative nature of working in industry and how delegating can empower people Chapter 3: How ...therapeutics at Novartis - listen to learn more about Crystal’s exciting career path, to gain insight ...metrics for success. The importance of thinking about the big picture and not just the next experiment...resources in our Mentoring for Scientists Series Learn about Turning Your PhD into a Career with Joanne Kamens...

...ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help keep...Journal of applied phycology27.6 (2015): 2321-2332. 3. Contamination, Ileana S., John P. S., Basic Methods...of the hood while work is in progress.Learn more about cell culture contamination in this post from the... bath constantly at 37°C for this purpose.Learn about the effects of CO2 on sample pH Cell morphology:... Set up a routine to split cells Don’t be lazy about splitting cells, instead try to form a routine. ...secretion of lactic acid (a by-product of cellular metabolism that is toxic to the cells), and need to be split...

... the lysosome where they are finally degraded (2, 3). Once you've created your fusion protein, it's important...2000. The Cell: A Molecular Approach. 2nd Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory Pathway...the context of the cell. However, much knowledge about functionality can be gained by understanding the...subcellular marker proteins, it’s possible to get an idea about the subcellular structures your protein resides ...with. These “colocalization” studies give insights about the proximity of two proteins within the same subcellular...Additional Resources on the Addgene Blog Learn More about Plasmids for Endogenous Gene Tagging in Human Cells...

...DNA primers that bind upstream (5’) and downstream (3’) of the DNA region of interest. Primers are crucial... the dNTPs.  Let’s jump into the specifics of the 3 basic steps of a PCR reaction:  Denaturation- To...If you have ever worked in a molecular biology laboratory you have likely done a polymerase chain reaction...uses ddPCR for AAV titrating. You can learn all about ddPCR for AAV titration and find helpful tips and...method that can help save time and effort in the laboratory.  There are two main categories of multiplex ...use a single pair of primers, like those stated above. Multiplex PCR is also used in high throughput SNP...

...Burgess, Shawn., et al. "Questions about NgAgo." Proteins & Cell (2016). 3. "NgAgo: Genome-editing by a DNA-guided...success of CRISPR-Cas9 across multiple model organisms (3) lies in the versatility of its delivery methods. ...technology. To dig more into what researchers felt about NgAgo, I conducted a survey and asked researchers...successful, and the second section asked basic questions about the NgAgo protocols used by respondents (transfection...PMC4343198. Resources on the Addgene Blog Learn More about NgAgo Enhance CRISPR Targeting Specificity with ...

...binds EQKLISEEDL (pronounced “equi-kli-seed-le”). (3) Tag all the things!   Myc1-9E10 and the Myc-...mapping functional domains of Drosophila hsp 70. Embo J 3:3087–3093. https://doi.org/10.1002/j.1460-2075.1984...Do you ever wonder about the origins of some of the common techniques or tools you use in the lab? Take...times of the myc tag.  Origins of protein tags For about as long as the disciplines have existed, cell and...examples of new versions of Myc1-9E10 mentioned above basically involve taking the Myc1-9E10 variable ...

...which is achieved by deleting a large portion of the 3'LTR in the transfer plasmid. The deletion prevents...number of HIV genes required for virus production (3 or 4). Third-generation vectors contain gag, pol, ...Plasmids 101 Learn about genome-wide screening with CRISPR and Lentivirus Read about the ClonTracr Library... the proper cell line is used; several common laboratory cell lines including 293 were immortalized with...

...be controlled by blue light (Bubeck et al., 2018). 3. Reduce cytotoxicity of CRISPR-Cas editing in tissues...Disabling Cas9 by an anti-CRISPR DNA mimic. Sci Adv 3:e1701620 . https://doi.org/10.1126/sciadv.1701620 ...Bondy-Denomy et al., 2018). For example, AcrIIA4 mentioned above indicates that it’s the fourth Acr protein discovered...Cas9 Variant for Your Next Experiment Read more about Optogenetic Control of CRISPR   Resources on Addgene.org...

...Ras pathway. The term “Ras” collectively refers to 3 proteins (H-Ras, K-Ras, and N-Ras) encoded by a family...cell 25.3 (2014): 272-281. PubMed PMID: 24651010. 3. Ledford, Heidi. "The ras renaissance." Nature 520.7547... cancer are yielding promising results in the laboratory, and Addgene is accelerating this research by...within that pathway that are of interest to their laboratory. A single mouse click on a gene of interest will...

...  Chromoproteins for allelic exchange Figure 3: pTOX plasmids expressing amilCP and tsPurple genes...genetic modification integrates into the genome (Figure 3). Here, the chromoprotein is expressed and gives the... Fluorescent Protein 101 blog series Learn more about FRET Find protocols and tips for making plasmid ...

...encapsulating the protein of interest (Figure 3).   Figure 3: Schematic representation of the MPyV system...acid, suggesting that MPyV system can improve metabolic pathway yields by encapsulating a single enzyme...

... Consulting firms deploy case teams, usually with 3-6 consultants, who work on analyzing the situation...the client to prioritize investments over the next 3, 5, or 10 years. Complicating these challenges is ...company to collaborate whenever it faces an important challenge. The types of projects mentioned above are just...and why you might enjoy doing it. I’ll also talk about the skills you’ll need to do well as a healthcare...often work with such teams to help make decisions about R&D investments in either new internal efforts, ..., or through acquisitions of or collaborations with other companies. Medical Affairs groups work on building...building awareness among physicians and scientists about clinical research, drugs, the diseases they can ...

...into Stage 3 or Stage 4 cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the...common cancers like lung (about 224,000 diagnosed this year) or breast cancer (about 246,000 diagnosed this...Mesothelioma Cancer Alliance. We have learned much about the causes of cancer and the different avenues that... asbestos exposure to the time of diagnosis) is about 35 years, but it usually ranges from 20-50 years... an orphan cancer; it is rare disease with only about 3,000 new mesothelioma patients diagnosed every ...from mesothelioma several years ago – recently collaborated with Senator Barbara Boxer to introduce the ...Additional Resources on the Addgene Blog Find out More About Cancer and Addgene's Cancer Collection Pages Learn...