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...might affect your work, and how understanding it can be used for good.  First, why are plasmids incompatible...replication. However, in iteron-containing plasmids,  RepA can also bind to its own promoter and inhibit its own...., 2015).  It is also worth noting that plasmids can be symmetrically incompatible, meaning that both ...the other, usually a smaller, higher copy plasmid can more effectively titrate replication or partitioning... of replication, the negative regulation system cannot distinguish between the different plasmids. The...to an identification issue: the CBPs in the cell can’t tell the difference between plasmids if they have...

...this post we'll walk through a variety of ways you can use AAVs to improve your genome editing experiments...pAAV-SEPT-Acceptor and pAAV-TK-Acceptor) so that they can be easily adapted to edit your gene of choice. These...CRISPR components as ribonucleoproteins (RNPs) that can be supplied separately from the viral vector. This... a variety of cell types, now you know that AAVs can provide a powerful boost to genome editing experiments...AAV gene editing to introduce naturally occurring cancer-causing mutations into the endogenous allele of...Professor of Oncology at the Lombardi Comprehensive Cancer Center, Georgetown School of Medicine. His research...consequences of cohesin gene inactivation in human cancer.     References 1. Russell, David W., and Roll ...

...research applications. Scientists around the world can upload AAV validation data, sharing what works and...community to maximize the positive impact that science can have on the world. ...

... graduate student life. Senior graduate students can sometimes be elusive or have a certain mystique that...our next episode, Niroshi and Ben discuss how you can leverage your graduate school community to help keep...beyond graduate school. Ben J. Vincent is a Ph.D candidate in the Program in Genetics and Genomics at Harvard...

...titer as compared to previous lentiviral vectors. It can be used both to knock-out single genes in clonal ...need help cloning a gRNA into lentiCRISPR v2, you can take a look at the protocol kindly provided by the...used for includes work probing mTOR signaling in cancer cells, deciphering the mechanism of matrix metalloproteinase...

... toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization of a protein of interest... tags? Short epitope tags like FLAG, HA, and Myc can adopt a number of different conformations and thus...antibody. From their initial pool of candidates, they selected 12 candidate protein domains from non-vertebrate...

...more concurrent projects in varying stages, which can get messy if you’re not on top of it. In a science-based...yourself with knowledge of software development so you can pick up the jargon and know the concepts as soon ... technology tools with the customer in mind. You can follow her on twitter @rgrubinstein. Additional resources...teams that run within the agile framework If you can’t find internships at science companies, try to find...themes and customer pain points we’ve identified. I can’t wait to send it out. 4:00 PM--Respond to customer...

...change rapidly during neuronal activity, scientists can use GECIs to watch neurons fire under the microscope... multicolor experiments. With OCaMP, researchers can image multiple brain regions or cell types at once...The Lohman lab has a limited quantity of AAVs that can be requested by contacting Alexander Lohman (alex.lohman...

...components are used to package lab grown viruses. It can be intimidating at first to look at so many plasmid...). VSVG is a broad tropism envelope protein that can also be pseudotyped to alter infectivity. Next, the...While not required for lentiviral production, they can be helpful for certain applications and aid in ease...

...serum There are a few different ways the antibodies can be isolated from the serum. Protein A/G purification... the immunogen. A different type of purification can be performed to isolate antibodies only recognizing...different epitopes of the target protein. While this can increase background staining (see next section), ...

...world by storm. Like RNA interference, these enzymes can knock down RNA without altering the genome, but Cas13s..., the Cas13d orthologs described in these papers can independently process their own CRISPR arrays into... require a protospacer flanking sequence, so you can target virtually any RNA sequence! In bacteria, Cas13d-mediated...efficiency; where shRNA treatment produced 500-900 significant off-targets, CasRx displayed zero. Unlike Cas9...

... sex of their cells when doing experiments—which can lead to failed research. For example, an international...cells and cell lines degenerate over time—and sex can be hard to determine. To remedy this, Shah et al.... been made in this area in recent years, and you can see a summary of this literature in our health and...of cardiovascular disease, 126,000 fewer breast cancers, and 145,000 more quality-adjusted life years. ...Shah et al., “Do you know the sex of your cell?” I can’t tell you how many researchers I’ve talked to who...the most commonly used cell lines appearing in American Journal of Physiology-Cell Physiology over the...product design. But eyes have been opened—and we cannot return to a world that ignores gender. Many thanks...

...for a curated list of coregulators). Coregulators can function as coactivators and corepressors depending...complexes, a member of a single coactivator complex can serve as a rate-limiting conduit to control the actions...and reagents in the field. Additional information can be found in the Datasets and Reagents sections of...of therapeutic indices, including hypertension, cancer, diabetes, cardiovascular disease, cholesterol ...

... template to Cas9 Homology-directed repair (HDR) can be used to make precise modification in genome editing...every 8 bp). Nme2Cas9 is also small enough that it can be packaged into AAV. The lab used single-AAV delivery...base editors Nme2Cas9 CRISPR interference in Candida albicans E. coli genome-wide inhibition library To...All-in-One AAV constructs CRISPR interference in Candida albicans A new CRISPR interference (CRIPSRi) platform...platform for targeted gene repression in Candida albicans was developed by Rebecca Shapiro’s lab. After testing...libraries also include 2,400 gRNAs without any significant hit across the E. coli genome as a negative control... fold and >3,800 fold in human cells with no significant reduction in editing efficiency. Read the paper...

...Anyone affiliated with the recipient organization can create an Addgene account and place an order: a purchasing... How do I pay by purchase order (PO) number? You can find answers to other ordering questions here. In...it on behalf of depositing institutions. Addgene cannot fulfill orders based on PO documents that are sent...

...Sometimes the PCR products can look smeared, so having an undigested control can help distinguish between.... Cas9 delivery Overview: Before any editing can begin we need our A549 cells to express Cas9. In ...Addgene viruses are provided at a specific titer, you can better plan your transductions and use a range of...well dish. My protocol with many additional tips can be found here. Figure 3: A surprise for the author... implicated in several human cancers) in A549 cells (a human lung cancer cell line), armed only with viruses... CRISPR, I was able to knockout BRAF in a human cancer cell line in about 1 month, which I’d say is pretty...

... complicated solution like cell lysate. But they can be a lot of work — and what if you want to detect... stripping is here! Rather than start over, you can remove the detection antibodies from your membrane...brittle, so proceed with caution. Unlike PVDF, they can be returned directly to buffer without a re-wetting...

...susceptible to redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols and ...change its fluorescence properties. The biosensor can also be used along other thiol-containing acceptors...further enrichment. Tian notes that this platform can be used to engineer many types of biosensors.   This...the brain. “We hope that iGluSnFR3 will enable significant steps forward in our understanding of synaptic... Brandán Pedre Pérez is a postdoc at the German Cancer Research Center (DKFZ) who has developed new redox...withdrawal Elizabeth Hubbard is a fourth year Ph.D. candidate at the University of California, Irvine where ...

... features we can offer online for Addgenies and to serve external customers. I also can’t wait to see ...DNA distribution to ensure scientists everywhere can access plasmids despite the evolving export/import...one of the main reasons I have so few worries and can often focus on the excitement of what we are accomplishing... employee pool and we get a lot of successful candidates from internal referrals. Our long reaching reputation...

...I ended up at pgEd, what I do there, and how you can set yourself on a path towards a similar position...turn crucial if we want to make sure that everyone can be informed and empowered to make their own decisions...ability to talk about science in a language that can be understood by those who are not experts in particular...