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...to a target polypeptide. Multimerization in the SunTag system occurs via antibody-peptide labeling; specifically...they righlty deserve, but, in this post, we'll provide a small sampling of the many amazing new plasmid...-2xNLS-VP64 and dCas9(N)-FRB-NES). This system provides users greater temporal control over CRISPR/Cas9...successful in regulating gene transcription using DNA-guided optogenetic tools; however, targeting the necessary...overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or Zinc Finger Nucleases...Nucleases, the Gersbach lab has modified the RNA-guided CRISPR-Cas9 system to create a tool that is quick...flexibility of the LACE technology makes this system widely accessible for many potential applications. Polstein...

...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 ...

...digest, PPIs are identified via mass spec analysis. Biotinylated proteins are considered possible interaction...distinct functions. Many existing methods for identifying PPIs, such as affinity purification mass spectrometry...spectrometry (AP-MS), lack the ability to specifically identify proteins that interact with a particular protein...the validation of a binary PPI as well as the identification of additional interacting factors. Problems...detect weak and/or transient PPIs. Many PPI identification techniques rely on protein interactors to stay... AP-MS. This requirement often prevents the identification of weak and/or transient interacting proteins...potential interactors with a bait protein but the flip side is that it’s difficult to know which proteins interact...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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 ...

...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...

...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 ...

...'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...

...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 ...

....  These primers are typically ~70 nucleotides long (20 nucleotides that anneal to the DNA sequence of... Frequency Special Considerations dsDNA Insertion or deletion of >20 nucleotides 1 out of 104 to 105...tube. The donor DNA substrate only requires ~50 nucleotides of homology to the target site for recombination...insertions or deletions greater than approximately 20 nucleotides, while ssDNA substrate is best for point mutations...of interest and 50 nucleotides of homology to regions flanking the target site).  The best applications...out of 104 to 105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability for ...substrates can be generated by ordering synthetic oligonucleotides or denaturing short PCR products. Either way...

...science policy even is! In this article, I hope to provide a brief explainer of the who, what, where, when...The folks who work in science policy come from a wide variety of backgrounds with technical expertise ...creatively connect seemingly disparate individuals and ideas toward solving one problem. It often requires thinking...the field.  Read the CCST Science Policy Career Guide to learn more about science policy as a profession...in DC, is that so much policy actually happens outside of the government itself. You can work to influence...communities. Think a university system who develops evidence-based university policy to combat sexual harassment...harassment in academia or a large company who decides how they are going pursue regulatory approval for a...

...easily and precisely identifies plasmid features that differ by only a few nucleotides, such as the fluorescent... BLAST to verify plasmids. Analyzer guide A quick guide to all of the tabs listed above and their...Feature Library - SnapGene’s software seamlessly identifies a variety of common plasmid features such as ... Enzymes, and Primers - The new maps have the identities of most features labeled directly on them making...making them much easier to quickly identify. Improved Restriction Enzyme Handling - SnapGene’s software..., & downloadable .gb & .dna files Once you’ve identified a plasmid that you want to learn more about, ...your sequence viewer of choice. If the depositor provided a custom annotated GenBank file, this can be found...

... Other useful backbone modifications to consider include amide linkages, unlocked nucleic acids, and a...to simply swap out several of the ribonucleotides for deoxynucleotides. A partially DNA gRNA is surprisingly...perfect guide; now check out our blog posts on choosing the best Cas9 enzyme to pair with your guide and ...larger CRISPR RNA (crRNA). In addition to the 20 bp guide sequence, the crRNA also contains a ~20 bp repeat... a constrained ethyl.  Can’t decide which RNA backbone modification is best? Studies suggest coupling ...use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications, specifically...photoactivatable and photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable...

...amino acids or peptide tags. 5) Strong protein-protein interactions with other peptides. Created with ...Addgene’s antibodies are provided in a buffer with the antimicrobial agent sodium azide, which can interfere...spectral properties of the fluorophores you are considering to make sure that they’ll be compatible with ...antibody conjugates (especially if you start to consider clinical applications). But there are a few other...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...multiplexing potential. You will find antibody-oligonucleotide conjugates used in assays like proximity ligation...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...

...Fab Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two ...two identical heavy chains and two identical light chains) each with a constant region and a variable region...chains are covalently linked to one another by disulfide bonds between the two heavy chains and between...four chains make a Y-shaped protein that can be divided into the Fragment Crystallizable region (or the... means that it is relatively easy to physically divide up the different regions, both through genetic ...proteolytic digestion that retains an antibody’s disulfide bonds within the hinge region. The resulting product...')2 fragments, which reduces the hinge region disulfide bonds, resulting in two independent Fab fragments...

...in the face of evidence” (4). Frustration among scientists has been rising as evidence of climate change...not in the job of having evidence”, scientists want to make sure having evidence is the job of everyone ...marching for every cause. The references provided also only provide a snapshot into the opinions or research...Health endanger scientists’ ability to uphold their side of the bargain and do the research required to inform...while alienating the other While scientists do identify as Democrats more often than Republicans, the ...Republicans (16). This is a Pro-Science and Pro-Evidence march. Who is marching and why? Who? All sorts...include science in decision making, it will be considered a success. Below are resources for the march ...

...mutagenesis of Spinach. In doing so, they identified the nucleotides that contribute to the instability of...describes the enrichment process used to evaluate oligonucleotides for binding affinity to a specific target,...molecule derivative of GFP called 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). DFHBI is membrane ...studies. Additionally, Spinach, the first aptamer identified to bind DFHBI, is highly unstable in vivo due...Spinach. It was developed in a study of artificial guide RNAs that can be used to simultaneously target cellular...Broccoli, the other green fluorescent aptamer, was identified through directed evolution. First, a pool of ... that activate the fluorescence of DFHBI were identified using systematic evolution of ligands by exponential...

...previously induced gene knockout. Besides amine- and DNA-binding dyes, nucleoside analogs like bromodeoxyuridine... useful for classifying and sorting cells by identifying lineage markers, like many of the cluster of ...one or several of those techniques can be used alongside standard surface labels. Here, I will introduce...streptavidin conjugates are commercially available for a wide range of fluorophores, allowing for great flexibility... you to include further (non-lineage) markers alongside, e.g., for cellular activation. In addition, FACS...interest is not located on the cell membrane but inside the cell. Due to their chemical nature, antibodies...antibodies to enter the cell (Figure 3). A useful side effect of the fixing/permeabilization (fix/perm)...

...attribution: Zetsche, Bernd, et al. "Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. Cpf1...through homology-directed repair. Both Cpf1 and its guide RNAs are smaller than their SpCas9 counterparts,...smaller than any of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the... al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs known to...orthologs was only slightly lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with... with Cas9 orthologs, Cpf1 efficiency varies widely with gRNA sequence. Both groups used multiple methods...

...they can’t be avoided. What are off-target effects? The CRISPR-Cas system is an RNA-guided nuclease that... gene expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often... concern, or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...off-target measurements at those sites to get an idea of how high the off-target frequency is (more on...many more. While such software typically only considers DNA sequence and doesn’t take into account chromatin...the genome to reduce off-targets, it is time to consider your Cas enzyme. Even if there are similar sequences...

...donor oligonucleotide can be electroporated into the mouse zygote (Qin et al., 2015).  When inside the ...However, if repair material is provided (in the form of an oligonucleotide or plasmid), precise changes ...genome, the mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would...for genome editing in eukaryotes, with the most widely used CRISPR genome editing system derived from ... CRISPR/Cas system makes use of 3 components, a guide RNA (gRNA) of about 125 nt that specifies the target...break (DSB) at the target site, and a donor oligonucleotide or plasmid as the repair material if needed...create a mouse model, the gRNA, Cas9, and donor oligonucleotide or plasmid components are brought together ...