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...go wrong. We hope that we have provided you with a few ideas to consider if you do run into a roadblock... faced with a roadblock. In this post, we will provide an overview of some of the common challenges associated...such events, newer lentiviral packaging systems divide the viral elements among multiple plasmids. Second...packaging systems please see Addgene’s Lentiviral Guide. While the third generation packaging systems offer...experiments a bit more challenging. With these considerations in mind, when planning an experiment users ...transgene, and downstream applications should all be considered when choosing the correct system. Recently, ...transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for larger ...

...microscopy techniques   Fluorescent Widefield Microscopy Fluorescent Widefield Microscopy is the most common...to solve two drawbacks of widefield and confocal microscopy. First, widefield and confocal microscopy project... visual system relies primarily on contrast to identify individual objects and perceive the world around...When performed correctly, fluorescence microscopy provides a high contrast image in which a bright signal...contrast, color contrast, to an image and thereby provide molecular or structural specificity to the viewer...microscope objective evenly illuminating the entire (wide) field of view. Fluorescent light traveling back...one time, limiting photobleaching. Similar to widefield, the entire field of view is excited at one time...

...Here, we will provide an overview of how to create and use a standard curve and provide some general considerations... considerations for scientists planning to use them in their assays.  What is a standard curve? Scientists...serial dilution of a control protein, incubated alongside an unknown sample with the BCA reagent. The absorbance... a known control sample, called a standard. The ideal standard curve has at least five dilutions, with... the data points generated by replicate values provides useful information about the accuracy of the curve... must be included every time the assay is run, ideally with freshly prepared buffer. This is because standard...users aim for an R2 above 0.95.    Additional considerations When running assays that rely on a standard...

...signal-to-noise ratios in two-photon imaging, making it ideal for video recordings of large populations of synapses...screened a large library of iGluSnFR3 mutants, identifying the two  best-performing variants as iGluSnFR4f...vector that contains everything needed to replicate inside a host cell but with space to insert your genetic...and strong expression after inserting EGFP and provide step-by-step protocols for viral generation and...retrotransposons for DNA editing By Emily P. Bentley For RNA-guided genome editing, think beyond Cas9 with STITCHR:...new site-specific retrotransposon elements they identified and characterized (Fell et al., 2025). In retrotransposon...that moving the RNA untranslated regions (UTRs) outside of the homology elements allowed for scarless insertion...

...newer lab members. You must get an idea of what to expect and decide if it is what is best for you. Some...development? To get a better idea of whether or not a lab head is a good role model, consider questions like: How...publication record show evidence of industry collaboration? If you are considering a career at a small, ...focused on choosing a lab, but almost all of these guidelines can apply to any workplace or job. It amazes ...sections below. Mentoring relationships can help identify your priorities and needs, but sometimes it just...talk to as many lab alumni as you can or schedule video/calls if they are not local. Alumni meetings can...Former lab members can give you a fuller picture. Consider talking to people in the lab across the hall or...

...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...

...experiment - there are a number of outside factors that need to be considered as well. In this post, we’ll ...overnight, so mark the vial, add the incident to your records, and consider the following questions as you ...ll touch on antibody storage, buffer considerations, and give just the lightest of nods towards conjugates... and illustrious lab career, you will likely accidentally leave your antibody out on the bench overnight...1: Blugene fading into darkness because they accidentally left their antibodies out. It's okay, Blugene...could affect the reaction. For instance, sodium azide, a common antimicrobial agent added to buffers, ...reaction. Many commercial antibodies come with sodium azide already included in the buffer, which will be noted...

...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...

...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...

...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 ...

...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 ...

...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...

...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...

...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...

...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 ...