...common, besides being one of the five senses? GPCRs or G-protein-coupled receptors (GPCRs)! Aside from these...molecule ligands such as clozapine-N-oxide (CNO). DREADDs are ideal for chemogenetics as they are relatively...quick guide to chemogenetics Resources on Addgene.org Check out Addgene's chemogenetics guide Find chemogenetics...with heterotrimeric G proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins ...Gα), beta (Gβ), and gamma (Gγ). G proteins are divided into four subtypes, based on their Gα subunit: ...that activate many GPCRs are still unknown. To identify some of these unknown ligands and their GPCR binding...Roth lab showed that this platform accurately identified 120 known GPCR targets demonstrating its potential...

...the plot that divides it into four, using the location of the cell populations as a guide  (Figure 3.) ...machine that everything inside the gate is one population and everything outside the gate is a different... It can be used to measure protein expression, identify rare cells, or even sort out single cells through... to use increasingly more stringent markers to identify your specific cell population of interest. The...reads/interprets the signals, the axis is instead divided into positive and negative signal at a point on....      Figure 3: It's often easier to identify different populations by imagining (or drawing...roughly group the cells by size. This is done using side scatter (SSC) and forward scatter (FSC), which measure...

...transcriptase. The Cas9 nickase is guided to the DNA target site by the pegRNA — a guide RNA that also encodes the... 75,000 pathogenic genetic variants have been identified in humans and cataloged in the ClinVar database...developed in David Liu’s lab in 2019, has added considerably more precision and flexibility to the CRISPR...does it work? First, an engineered prime editing guide RNA (pegRNA) that both specifies the target site...editors are preferred. For instance, if target nucleotides are positioned within the canonical base editing...HDR) stimulated by double strand breaks has been widely used to generate precise changes. However, the ...optimized for each application, and the efficacy varies widely — it still almost always results in higher ratios...

...to an oligonucleotide depending on your downstream application. Biotin labelled oligonucleotides can be...attached to a photoreactive arylazide group. Under strong visible light, the arylazide group becomes reactive...in the small intestine from the microflora that reside there. Biotin is a cofactor that in its active ...Avidin is a glycoprotein that consists of four identical subunits that can each bind one molecule of biotin...macromolecules it is added to. Biotin also has an exposed side chain that can be easily manipulated to make reactive... biology and biotechnology purposes.  Once a polypeptide is biotinylated (more on this below) it can be...multiple biotin binding sites found on avidin are also ideal for signal amplification during these experiments...

...Experimental guidelines Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the...PCR process, you need a high-fidelity polymerase. There are many high fidelty polymerases on the market;...technique that can be used to introduce specific nucleotide substitutions (or deletions) in a tailored manner...detects primer multimerization (purple) which is evident by an increase in the size of the lower band (portion...the plasmid of interest during the PCR reaction. Ideally, your primers should be free of palindromic and...Methylation deficient bacterial strains can be identified by the dam13(-) mutation – you’ll want to steer... exonuclease activity (increases amplification fidelity), and no 5’->3’ exonuclease activity (which could...

...there, then start identifying the resources needed to accomplish that goal. 2. Identify the tools in that...past year to get an idea of how much you can expect to use in the future. 3. Identify the skills needed ... clients focus on their experimental needs and consider a budget as a resource checklist. A budget is ...Fluorescence Scopes, Flowcytometers, etc.). After identifying all the major equipment, categorize all of your... needed to do your science. This will help you identify the talent pool that you’ll need to tap for your...materials, and resources that you’ve thoroughly identified. Do online searches, and seek advice from your...salaries in Kansas versus salaries in NYC will vary widely. Therefore, start up awards should reflect salaries...

...yellow-light sensitive chloride ion pumps, activation of which results in efflux of chloride ions, subsequent...the injections must be near identical for all of these surgeries. Identify coordinates through a rodent... if you are not familiar with it, check out the video below). That’s not to say that the technique doesn...methodological and experimental limitations and considerations? I recently spent two months in the lab of ... at NIDA and some of the practicalities and considerations you may not find in the materials and methods...use optogenetics The most important question to consider is what exactly do you want to use optogenetics...are also available. Picking your opsin  You must consider whether you wish to stimulate or inhibit neuronal...

...because they are inside the virus itself. While these impurities are generally considered innocuous for ...tool that can be used to identify DNA contaminants in AAV preparations and provide information on these DNA...NGS) of the viral genome to confirm viral genome identity and serotype. Additionally, we check viral transgene...multiple vectors in parallel because the risk of accidental errors increases. In addition to human error,...helper plasmids or the cell genome can be packaged inside the vectors during production. These impurities...NGS at Addgene to sequence all the DNA packaged inside the viral particles (Figure 1). Briefly, packaged...Raw sequencing data is analyzed to determine the identity and serotype of the packaged DNA and look for ...

...addgene.org CRISPR pooled libraries Guide to pooled libraries CRISPR guide AAV guide ...expression. Episomes are circular pieces of DNA that reside outside a host cell’s genome. In this state, AAV cargo... for each experiment. Additional considerations One of the downsides to using AAV is the limited packaging...valuable part of the molecular biology toolbox to identify new target genes for drug discovery or to understand...effort. The versatility of Cas effectors allows for a wide variety of screens, including interference, activation...rely on lentiviral systems, a fact supported by the wide array of lentiviral-based CRISPR libraries available...selection). Next-generation sequencing (NGS) to identify genes of interest. Figure 1: Overview ...

...and between genomes, and have been adapted for a wide variety of applications - as mutagenesis tools for... 2019; Strecker et al., 2019), which direct RNA-guided transposition. By natively combining the DNA integration...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) by the Sternberg group, ...system. The four major components required for RNA-guided DNA integration are (Fig. 1A, top):  A CRISPR ... CRISPR-Transposon system. (B) Mechanism of RNA-guided transposition in type I-F INTEGRATE. The requirement... are also efficiently recognized by the system. Guided by the crRNA, QCascade complexes survey the genome...and C). While requiring fewer components for RNA-guided DNA integration, Type V-K systems frequently produce...

...the CRISPR toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a ...cleaving CRISPR nuclease Cas13a was originally identified in 2015 (Shmakov et al., 2015). They were using...acquisition following infection, as a form of “bait” to identify new CRISPR-associated proteins within the bacterial...bacterial genome. From this analysis, they identified 53 potential candidate genes that fell into 3 categories...Common CRISPR Enzymes Name  Enzymatic Domains Guide RNA Target PAM (PFS) Cleavage Mechanism Cas9 HNH...requires that each DNA target have high sequence identity to the spacer sequence and contain the appropriate...Cas13a as a diagnostic tool. A pool of DNA or RNA nucleotides containing a sequence of interest (red) is amplified...

...genes we’ve tagged are widely recognized markers of cellular structures, we provide guidance on how to make...in creating the plasmids was to identify the gene of interest and decide which terminus, N or C, to tag...structure of interest. Endogenous gene tagging is an ideal solution because it allows for tagging and visualization...plasmids that use CRISPR/Cas9 to endogenously tag a wide variety of genes with GFP. These plasmids are available...plasmid that contains long stretches of DNA on either side of the fluorescent protein sequence that is homologous... being inserted: one thousand base pairs on each side, in fact. Later, when we transfect the cells, these...GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone...

...Viral vector topic page Beginners guide to viral vectors Quick guide to all things lentivirus Resources...Resources on Addgene.org Gamma-retrovirus Guide Lentivirus Guide ... or miRNA. The ever expanding CRISPR field makes wide use of retroviral delivery systems, especially lentiviral...functional screening of thousands of genes in genome-wide experiments. Gamma-retroviral vs. lentiviral vector...gamma-retroviral vectors in our Gamma-retrovirus guide and find popular packaging and gamma-retroviral ...Lentiviral Systems can be found in our Lentiviral guide. Popular packaging and lentiviral envelope plasmids...VSV-G, may be used across both systems. Below I provide a brief overview of a few popular gamma-retroviral...

...this post to guide you through them and make the best use of all the information provided by your colleagues...depositors have provided primer sequences for use, these are listed.  We also provide a link to our list...this plasmid from our search page (check out this video for a walkthrough of the ways you can search our...flames about? Flame logo: The flame that appears beside some of Addgene’s plasmids is an indication of ...their own labs. We highly encourage depositors to provide plasmid maps whenever possible. GenBank files can...research! Sequence information: Depositors may provide full or partial sequence information for their ...reactions (‘Addgene Sequences’).  These sequences are provided in FASTA format (example) and can be analyzed ...

...2020 by Marcy Patrick. CRISPR technology has been widely adopted for genome editing purposes because it'... your next experiment! In this blog post we’ll provide an overview of some CRISPR mammalian expression...and potential delivery methods.   General considerations for planning your CRISPR experiment As with...experiment, there are many factors that need to be considered during the planning process. For CRISPR experiments...choose between them? One of the first steps is to identify what CRISPR components you will need to deliver...deliver a donor plasmid or single-stranded DNA oligonucleotide, so you will need to make sure your expression...are compatible with all your components. Next, consider the best form for those CRISPR components based...

...wasn’t exactly my idea of a dream job, but I knew that I enjoy helping people and decided to compromise and...also explore a range of alternative careers outside of acidemia to have the best chance of finding their ...variety of different organizations, so I tend to provide them with an overview of my experience and how ...about the different positions to enable them to decide for themselves.  Here is my experience looking ...language in Spanish or French, and so they were ideal candidates for these positions.  I had to reassess... to university to study a Masters of Science I decided to apply to study a Master of Science (MSc) in ...research project was hard; I was researching the epidemiology of breast cancer and I loved the practical aspects...

...can also be exciting! Identifying a host institute or lab So, you’ve identified a gap or need in your ...collaborations Transferable Skills Guide: External collaborations Transferable Skills Guide: Teamwork      ...or maybe both, a visiting scientist stint may be ideal for you. Here, we will chat about who might want...visiting role is because the host lab or institute provides something that you can’t obtain at your current...in. Visiting a lab that has this expertise will provide you with guidance and training so that you yourself...new institute and mentor that they may want to consider for future work placements. The chance to learn...your research and decided you want to pursue a visiting position. What now? First, you’ll need to find someone...

...'s a good idea to bounce your ideas off of senior faculty members who can help you identify overly ambitious...the central idea. It should be at a high level, broadly understandable to scientists outside your field...least a few times with people that are outside your lab and outside your field. If they can understand and...?" is an open-ended list of proteins. Instead, consider "how do proteins X and Y regulate the Z pathway... or your data favor a different model. Finding evidence for or against a model is a standard scientific...coherent unified story and model can help make your ideas easier to understand and more exciting. Practice...that is easily visible from the back of the room. Consider going beyond the whiteboard. You may wish to create...

...some 1010 oligonucleotides could be screened for their affinity to a wide range of non-nucleotide targets...over the course of the next two decades, these nucleotide-based ligand binders would prove to be highly...their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells (even...such as a protein. Such flexibility makes aptamers ideal candidates for biosensors. Aptamer biosensors are...several different ways. Researchers have used oligonucleotides in fluorescent visualization techniques for...relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity, which is...- bypassing translation altogether with an oligonucleotide-only sensor could save precious minutes in ...

...why we decided to do an AMA in the first place and give you some reasons why you should consider using ...hope I was able to provide some good resources to the basic questions and to provide a small bit of support...interested in selling a product than communicating your ideas with people or discussing science, it is a great...users can participate in forum discussions about a wide variety of topics. Individual forums are hosted ...STEM, work-life negotiation, and mentorship, we decided to have her participate in an AMA on these topics...substantial traffic that we thought it would be a good idea. After a bit of research, we realized that the r...You can check out the r/Science AMA Submission Guidelines here, but essentially we just needed to send ...