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...String assembly gRNA cloning (STAgR) String assembly gRNA cloning (STAgR), developed by Stefan Stricker...developed another AAV toolkit - this time using the Staphylococcus aureus Cas9 (SaCas9). This AAV-based genome...high numbers of gRNAs in one overnight reaction. STAgR is highly customizable as vector backbones, and ...chosen. Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows CRISPRi...from various environments. It has the advantage of stable genomic integration and is easily transferred via...

...fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection...uninitiated, contains much of the information needed to understand how to use it. TLDR: This is a versatile mammalian...ribosome to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the ..., which had a point mutation in the puromycin resistance gene that made it less effective in certain cell...nickase), which nicks DNA rather than creating double stand breaks and may better facilitate homology directed...

...Lentiviral vectors have been a staple in molecular biology for over three decades. Widely used across...for a variety of research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for... isn’t so great. In this post, we’ll cover how standard lentiviral vectors work with regards to integration... risks associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can also...insertional mutagenesis while still allowing for stable expression of the transgene. This makes IDLVs an...integrase-deficient packaging plasmid instead of the standard packaging plasmid. For more details on how to ...., Adams, S., Thornhill, S. I., Parsley, K. L., Staal, F. J., Gale, R. E., Linch, D. C., Bayford, J., ...

...voltage response, brightness, and photostability. It enables sustained deep-tissue imaging in vivo. Image... and increased brightness, sensitivity, and photostability under two-photon illumination than previous...pAAV-EF1a-DIO-JEDI-2P-Kv-WPRE   Liu Z, et al. Sustained deep-tissue voltage recording using a fast indicator...ZAP70 (ZtSH2) which binds only the phosphorylated state of an immunoreceptor tyrosinase-based activation...options for potassium biosensors and are a promising starting point for further engineering. Figure 5: Structure-guided...lifetime imaging. B) LiLac responses are highly resistant to pH changes. Image adapted from Koveal et al...

...facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template...cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage. Summary of Site Directed...and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are... pieces of double stranded, circular DNA with staggered nicks, recent data (see Xia et al., 2015) suggests...products into will efficiently patch up the DNA. The resistance marker from the parental plasmid provides a mean...overhangs and ligate these blunt ends together in a standard ligation reaction resulting in a frame-shifted...Breslauer, Kenneth J., et al. "Predicting DNA duplex stability from the base sequence." Proceedings of the National...

...based at the University of Queensland, whose outstanding marine photography has attracted much attention...what science is about. For me it is essential to stay in touch with the living organism I study, to be...So photography can be used as a way to better understand and appreciate the organism, for both scientist...Geographic and Smithsonian Magazine, where the editor stated “Albert G. Richards, a teacher of dental radiography...on teeth, but on blossoms, thus revealing the startling inner beauty of familiar flowers." This new x-...departments to showcase the work of students and staff by holding scientific art competitions where creations...

...frustrated and no closer to starting your experiments. Get organized now! Start by gathering that pile of...wake up and get to the lab earlier, to read that stack of papers on your desk, and to tackle some new experiments...or projects) to make it easier to know where to start your search. Then identify the most frequently used... and resource investment required to change the status quo. But with recent improvements to ELN and online...ways to share your documents with your colleagues. Start small by identifying the protocols, guides, or references...

...conventional microscopes, amplified staining methods, and oligonucleotide-based staining. Of course, if you’re interested...multiplexed tissue imaging method that uses iterative staining and chemical bleaching to allow for high-resolution...reagents that you know work…preferably before you start your experiment. (And you’re certainly not alone...Biomolecular Atlas Program (HuBMAP) antibody metadata standards. And the dataset is rich. Experimental data...seeking to create an iterative process that is constantly becoming better, with more data, more discussion...

... from an analysis of human metastasis cohorts and a prior mouse metastasis CRISPR screen. The Chen Mouse... Using the structure of CasX at eight different states of assembly from cryo-electron microscopy, the ...mutated human estrogen receptor. This protein fusion stays in the cytoplasm, but with the addition of 4-hydroxytamoxifen...knockout libraries for cancer research. The Mouse Metastasis CRISPR-Cpf1(Cas12a) Double Knockout Library is...tumor suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double Knockout Library Find the...

...continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around...optimize this system for gene transfer, studying the stability of modified Ti plasmids during plant infection...versatile binary Ti vector for both transient and stable gene expression applications in plants. This smaller... To build a better binary vector, Sahoo et al. started with a vector commonly used by their lab, the 13...variety of plant species. The kanamycin antibiotic resistance gene is controlled by a weaker constitutive Nos...expression vector for transient assays as well as stable expression of foreign genes in plants." PLoS one...

...just dive right in and start injecting your virus, a successful AAV experiment starts with validation and...your system that can affect expression, before starting the selection process. Factors to think about ...expression.   Once you know that, you’re ready to start selecting, testing, and optimizing three key variables...serotype that works great in a GABA neuron, for instance, may not transduce the adjacent norepinephrine...in your system and then test each vector before starting your experiments (Rolland et al., 2016; Su et ...selection …is truly validation and optimization! Understanding serotypes, promoters, and titers will allow ...

...AAV.  Where do you start? Plasmids of course! You just need three plasmids to start making the AAVs you...endonuclease domain. Following replication, Rep78/68 stays attached to the AAV genome and docks with the genome...Alternative splicing and differing translational start sites generate the three VP proteins. The three ...clearly defined yet. Now you’ve got a basic understanding of the AAV RepCap plasmid! Check out this blog.../doi.org/10.1007/s40259-017-0234-5  Büning H, Srivastava A (2019) Capsid Modifications for Targeting and...

... of the project. The state of research is constantly shifting, and long-distance collaborations are becoming...between each meeting, but short enough that everyone stays on the same page. Ideally, these meetings include...Conference in Frankfurt, Germany When funding and circumstances permit, think about having collaborators spend...communication within an existing collaboration, or start something new! Additional resources on the Addgene...

...new to plasmids may need some extra guidance to understand the specific components that make up a plasmid...and techniques, and empower you with a firm understanding of the fundamentals. Our mission is to curate... more functional benefits to the host such as resistance to antibiotics, degradative functions, and/or...advantageous for survival, such as an antibiotic resistance gene. In contrast, plasmids utilized in the ...recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing... obtained. Selectable Marker The antibiotic resistance gene allows for selection in bacteria. However...

...process One major challenge when we started was creating a LEGO stage that resembles a real laboratory. ... pathways just waiting to be discovered. As we started building, we thought – how can we make the connection...in a custom made, colorful laboratory. When we started, the hardest part of the process was to embed personality... trained to follow protocols, guidelines, and standards. Bricking science reminded us of our inner children...children who have no preconceived obstacles, optimistically grasp at limitless potential, and who sometimes...

...was using.  I started to do little things to save reagents and money.  This was the start of my interest...resources where possible.  At the lunch table, for instance, we have a plastic container problem…reusable ... a popular topic in the media.  At one point I started to become very aware of how little care I took ...time and money. My other stingy science project started when I realized I was pouring large agarose gels... a small portion of the gel.  What a waste?  I started looking for smaller and smaller gel apparatus until...

...towards a career in science communication, an established field of inquiry with which I was not yet familiar...Herein, a good science communicator—with an understanding of how to appeal to a given audience and how... a scientific background and bring with them established interests and areas of expertise. Perhaps the...individuals respond to issues of unsettled science. Understanding just how the public utilizes insights gleaned... ours reflect a growing effort to address this obstacle in its many forms. A short stroll from my office...by the day, it has never been more crucial to understand just how we receive, process and respond to that...

...Tchetchelnitski V, Mehmet H (2012) Remarkable stability of an instability-prone lentiviral vector plasmid in Escherichia...culture, only the plasmid with the antibiotic resistance gene and origin will replicate, and the transgene...recombined vector backbone contains the antibiotic resistance gene (AmpR) and origins of replication (ori and...Recombinase-deficient strains like Stbl2, Stbl3, and NEB stable E. coli reduce the rate of recombination in samples...CS, Esposito D (2007) Improved recombinational stability of lentiviral expression vectors using reduced-genomeEscherichia...

...shipping staff agreed to come in and start getting plasmid deposits into the collection and to start shipping...closure was earlier than most companies. Since the start of this crisis, we’ve been very responsive to the...Addgenies home.  At the same time, as Addgene started dealing with this new reality of working remotely...processes are entirely self-supporting. We hire more staff as we need them to meet demand and to serve the ...

...stop you. Where do you start? Turns out like all good experiments, making AAV starts with some plasmids. .... You just need three plasmids to start making AAV:  the packaging plasmid which contains the structural...between the ITRs is ~4.4 kb. ITRs are inherently unstable due to their secondary structure, palindromic ...palindromic nature, and high GC content. This instability can cause partial loss of an ITR during routine plasmid... Plasmid video!   Now you’ve got a basic understanding of some common AAV plasmid elements! And while...