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...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the...use of both when making plasmids. Use repositories Repositories such as Addgene help share materials quickly...After the great Plasmid Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid...for the simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase.... This cost varies hugely based on a number of factors, but we can assume it’s at least equal to the $155...in a 96-well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any...any of the numbers in this post is a mathematical story well beyond my ability to tell. But even from these...

...knock out plasmids? Check out our Cre vector and Murine LoxP vector.   Turn it off for a little while Transient...? Check out our Retro expression vector and Lenti expression vector. Turn it on for just a little while...CRISPR for that, too! CRISPR activators (CRISPRa) are transcriptional activators fused to a dead Cas9. The...out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time If ...Addgene's Lentiviral dTAG cloning vector and SMASh degron tagging vector! Destabilizing domains Destabilizing...increase expression. There are several flavors of activators which can be used with this approach - read more...them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently...

...begun to use more precise genetic tools: activators, inhibitors, sensors, recombinases, and more. With ...by using cis regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational...Addgene blog Download the viral vectors 101 eBook Read our viral vector related blog posts Learn more about...With the help of adeno-associated viral vectors (AAV), neuroscientists can combine classical genetic tools... how these changes affect the phenotype. Viral vectors and genetic approaches are continuously refined...Education and Development workshop aimed at brainstorming strategies for using combinations of AAV-based...designing experiments with AAV: 1. Getting your viral vector into your cell of interest: AAV tropism and route...

... of stimulation to receptors. Optogenetics offers better control over these factors as the light source...more about optogenetics New to viral vectors? Browse our Viral Vectors 101 blog posts Learn more about precise...Addgene.org Browse all viral vectors available at Addgene Visit our viral vectors guide Find our chemogenetics...different advantages and limitations.  The four main factors in which chemogenetics and optogenetics differ ...offers a high degree of temporal resolution as receptors can be turned on and off simply with the presence...diffusion of drugs throughout the body, the onset of receptor activation takes minutes. As a result, it is not...response or track the exact time of activation. Receptor activity is also drawn out as drugs can remain...

... they monitor. Gene traps Gene traps are promotor-less constructs that contain splice acceptors. They...below), but fundamentally the reporter construct monitors the expression of a nearby gene. The reporter ...high/low) and patterns (tissue/timing) can be monitored under various conditions without necessarily modifying...stage you could infer that the gene which GFP was monitoring was being expressed in the same pattern. These...throughout an organism’s genome. These reporters have historically been used extensively in fly, mouse, and yeast...result but will still allow for expression pattern monitoring. Enhancer traps Like promoter traps, enhancer...traps, it will still likely disrupt the gene it monitors due to transcriptional fusion.   Examples...

...inheritance. CRISPR activators and repressors are thought to be reversible once the effector is inactivated...promoters. Other researchers additionally fused a TAL effector to LSD1 histone demethylase to demethylate enhancer...methylation and activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available...and repair pathways work downstream of Tet1 to restore the DNA after cytosine removal. Recently, Albert...alone, or coupled with DNA oxidation and repair factors that allow for increased gene activation at the... targeted enhancers. Although the lab used this vector in chick embryos, it also functions in mammalian...and this fusion allows for packaging into viral vectors and targeted delivery into hard-to-manipulate cell...

...bacterial prey. Currently, receptor binding proteins (RBPs, the host recognition factors of phages) constitute...Therefore, in addition to their utility in the health sector, phages represent a reservoir of carbohydrate binding...function of digestion time. For phage DNA that is refractory to enzymatic digestion, however, a physical fragmentation...a hose to a nitrogen gas tank with a pressure regulator. Other methods for physical shearing exist as ... blunt ends) and ligate into a bluntly digested vector designed for protein overexpression in E. coli,..., such as pET30a. If working with DNA that is refractory to enzymatic manipulation, amplifying phage DNA...primers bordering the multiple cloning site of the vector. In order to reduce the chances of some clones ...

...Existing methods for AAV quality control Historically, AAV vector QC has been sparse including only titration... procedures and Python scripts with laboratories and viral vector cores routinely producing AAV. We believe... our viral vectors related blog posts Read our blog post on AAV titers Browse viral vectors protocols ... platform to accommodate adeno-associated viral vector (AAV) samples and created a simplified process,... of quality control is sufficient but for viral vector core facilities producing a large number of viral...demonstrated that NGS is an effective tool to confirm AAV vector genome identity. These studies used SMRT sequencing... simplified approach, we sequenced over 100 AAV vector preparations and showed that VGS can quickly and...

...Louis provides a combinatorial indexing strategy that simultaneously tracks clonal history and cell identity...embryonic fibroblasts (MEFs) into induced endoderm progenitors (iEPs), a self-renewing cell type that can differentiate...iEPs by retroviral expression of transcription factors Foxa1 and Hnf4α, a process that like other reprogramming...CellTagMEF), at the end of the three-day transcription factor reprogramming treatment (CellTagD3), and 13 days... Morris Lab found that cells took one of two trajectories: a route towards successful iEP conversion or... The CellTags revealed that cells with common ancestors tended to have similar reprogramming outcomes....2019). Key takeaways of CellTag CellTag is a combinatory cell-indexing tool that combines scRNA-seq and...

...and money producing virus from select vectors in the repository yourself, you can now order ready-to-use...As some of the most popular lentiviral vectors in the repository are used to produce CRISPR tools, we’re...Expanding the viral vector knowledge base: You can help! We’ve been expanding our viral vector educational resources... production process while still ordering viral vectors. Virus production can be a frustrating process ...delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select gRNAs, and CRISPR pooled...expression of a gene under the control of the tet operator. AAV AAVs are the workhorses of both chemo- and... work with virus. You can find all of our viral vector blog posts here. If you have virus expertise you...