...overhangs with the same three nucleotides in a row; (4) no more than two nucleotides in the same position; (...that high-fidelity reactions could be done using overhang sets that broke rules 3—5. Fidelity, it turns... design high-fidelity Golden Gate Assemblies and overhang sets: NEBridge Ligase Fidelity Viewer™; NEBridge...reactions. NEBridge Ligase Fidelity Viewer This tool allows a user to check fidelity of an overhang set by ...remarkably high 71% fidelity, while the 52-piece assembly of a lac operon dropped to 49% fidelity, with significant...be strictly followed in order to generate high-fidelity reactions. (Need a refresher? The five rules are... a single tube. Their assembly had a predicted fidelity of 71% (Pryor et al., 2020). And in 2022, using...

... requires that you provide two out of six forms of documentary evidence: Evidence of receipt of major...obtain permanent residence. The recently established case Matter of Dhanasar provides the three-prong ...field Evidence of original scientific or scholarly research contributions in the field Evidence of authorship... to convey general information only and not to provide legal advice or opinions. The contents of this ...advice on specific legal issues.  The permanent residence “green card” process is a necessary and often ...uncertainty associated with filing a permanent residence case with United States Citizenship and Immigration...national researchers who require visas, permanent residence or citizenship to continue their work in the US...

...defined peptides. Munro and Pelham’s first published peptide tagging experiment involved a peptide from ...with unrelated peptides that already had high quality antibodies available - aka peptide tagging.  Key ...bound to the mid-protein peptide, while three bound to the C-terminal peptide. One of these C-terminal... of these new antibodies and identified Myc1-9E10 and its target peptide as a suitable pair for protein...from the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino...could be quite small. However, substance P has an amide group added to its C-terminus post-translationally...critical for antibody recognition. As such, the peptide could only be added to the C-terminus of proteins...

...for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking. The...One 2013 Oct 7;8(10):e76117. Irreversible peptide-peptide ligation using SpyLigase Building off their...that promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against...scientists to use CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries... to browse CRISPR plasmids, watch informational videos, download protocols, and more! Looking for background... CRISPR technology? See our improved CRISPR-Cas Guide. Wang et al., Science. 2014. Jan 3;343(6166):80...

...libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined ...10-6 mutations per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of ...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...O., et al. "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window... changes to a DNA sequence can help scientists identify and/or facilitate the evolution of new phenotypes...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...

...even single mismatches within a CRISPR guide can prevent the guide from binding to the genome or prevent...thetargeting sequences of guides across 8 CRISPR libraries. Samples are divided into those of African ancestry...approximately 20% more single nucleotide polymorphisms (SNPs) that map to guides in the Broad’s Genomic Perturbation...accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’ genomes... CRISPR screens in DepMap to see if they could identify the ancestry of the cell lines (European, East...germline genetic variations. They then wanted to identify cancer-associated genetic dependencies associated...relationship between the genetic dependencies they identified and their associated ancestries. While there ...

...adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in ... these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at...cleavage with some of these peptides, no consensus has been reached on which peptide works best. Likely the...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...the specific genes cloned into the vector.3  2A Peptides To overcome some of the disadvantages of the IRES...between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the ...Proline.  The table below lists the four common 2A peptides employed by scientists. 2A cleavage is universal...

...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ... the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing...reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation of...BLAST search pages. At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...

...A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations...mutation), blocking the guide RNA binding site works well. However, since some guide RNAs may tolerate a ...end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred...repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the intended sequence...efficiency.   But what’s better: PAM-blocking or guide RNA blocking mutations? This depends entirely on...however we have also demonstrated the efficiency of guide RNA blocking mutations. So if the locus does not...while taking advantage of the improved efficiency provided by blocking mutations. Essentially, you:  Step...

...social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to provide informative...using the example of our Antibody Guide, which was published alongside our new antibody services in the... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise.... Since our guides are heavily used, our graphic designer made most of the Antibody Guide figures. This...topics for the Addgene blog? Or why we make certain videos? In this post, we invite you behind the scenes ...you, whether you realize it or not! Many of our ideas come from social media conversations, conferences... all of our frequently asked questions!    Identify needed content Let’s take a look at how we create...

... that you provided for each sample. Guide Target - This is the user-defined 17-23 nucleotide sequence ...sequencing files and provide basic information such as your sample names and guide sequences, and ICE will...which also includes our CRISPR knockout guide design tool, guide validation tool, and guaranteed editing... Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on...editing field by making experimental workflows considerably easier, faster, and more efficient than previous...examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing ...time or as a batch of hundreds, and indicate the guide RNA sequence you used. The ICE tool will calculate...

... more fully identify with and be inspired by the mentor’s successful example. For evidence of this, you...Valencia): “IMP showed me a wide range of professional opportunities that are outside of our typical boundaries... Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by its Director, Dr.... closer than ever (although not physically) the ideas and experience from talented professionals to their...together with the universities, a bright future. IMP provides matched one-on-one international mentorship, with...globalized world where individuals can exchange ideas, network, collaborate, and innovate internationally...offered by programs like IMP USA-Europe not only provide the specific technical and logistic support to ...

... particular, one slide of the presentation made a lasting impression on me. The slide showed a complex...approach is to identify an appropriate list of genes to target. Thus, it is always a good idea to keep a bioinformatician...Sweden. It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you...screens targeting a (long or short) list of genes provided by the user. This approach is useful for several...effective by world class CRISPR labs.  Check Out This Video to See How Green Listed Works at a Glance!  CRISPR...subsequently sequenced by next generation sequencing to identify which gRNAs are differentially expressed in the...to dissect a complex signaling pathway, and to identify what part of a pathway to inhibit to block the...

...joint. Silly ideas (mining local salt deposits to sell at farmers markets), and serious ideas (how to stop...for the future. From good ideas to a SynBio startup At some point, our ideas got pretty good. We started...journal clubs outside of work. Our scientific training was put to use as we explored ideas, planned out...we could do to make ourselves marketable to the wider world and occasionally thinking about how we could...establishment hiring system altogether. We had a lot of ideas about things we could do for work, and they weren... together we make a pretty good team. We have confidence that we can take on any challenge and quickly...marketable product. During this process, we had an idea that survived the literature review, survived technical...

...chain-terminating or dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string...Sanger sequencing uses modified nucleotides, called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl...added and when. Compiling the identity and location of a given nucleotide, a researcher (or a simple computer...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...Edward M. Southern (1), can be used to detect DNA identity, size, and abundance. If a scientist wants to ...gene mutation, southern blotting can be used to identify the presence of that mutated gene. In southern...One example of southern blotting being used to identify a gene mutation is the genetic test used for sickle...

...whenever applicable?Against this, consider the cost of an accidental release. Science relies on popular... DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate flanking homology...homology into a germline cell can create an RNA-guided gene drive. In principle, this event could happen even...available. Consequently, the best way to avoid accidentally creating a gene drive is also the easiest: don't...barrier protocols, they can reduce the risk of accidental escape to a negligible level. One major reason...publish the concept and likely capabilities of RNA-guided gene drives in the summer of 2014 (after extensive...hope was that anyone else who came up with the same idea or wanted to build a gene drive in a model organism...

...to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA provided that target...the components necessary to: 1. Bind to a guide RNA The guide RNA, which will be described in more detail...of the site targeted by the guide RNA. In the absence of either the guide RNA or a PAM sequence, Cas9 ...synthetic guide RNA or gRNA (sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its...into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). Throughout the remainder...Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After ...Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9...

...Producing stop motion videos is very time consuming; producing a 1-2 minute video takes an entire week ...with the images thereby making the videos more real. To give you an idea of the amount of work, see how many...’s gotten easier now that I identify with the main character in the video. We’ve grown attached to the...Dalila Cunha de Oliveira. Bricking Science is an idea built, literally, 'brick-by-brick' to introduce ...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera:...most people don’t get to experience this very human side to scientific research. Find Protocols Lab Tips ...was born. We chose the stop motion format for our videos as it is the most approachable for LEGOs. Stop ...

... development. Paula Videira: Congenital disorders of glycosylation Paula Videira is an Assistant Professor...been working with the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases...through the BeHEARD Award. Congratulations to Paula Videira, Michael McMurray, and Richard Gronostajski who...steps in the glycan biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the...) activity due to mutations in the PMM2 gene. Videira’s group recently reviewed and reported that PMM2...studied and understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to ...altered in PMM2-CDG. These studies are expected to identify new therapeutic targets/solutions and enable scientists...

...the bulk of the peptides identified to be from the target protein. This approach is ideal for validating...the sample was identified as your target protein, allowing you to proceed with confidence.  Share your ...experiments that you’ve been planning, it is a good idea to pause and make sure that your antibody has been... appropriately validated so that you can have confidence in your results. But what is appropriately validated... This post will go over some of the general considerations you should review before using an antibody.... that your antibody is not binding to proteins outside of your target protein. Show suitability for an...There are a number of resources available that provide in-depth discussions on strategies for antibody...

...If you have a wide variety of protein amounts on the same blot, you may want to consider using a different...online/ask an expert Review protocol videos for a visual guide to good technique High background ...lightly marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and... buffers and strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing...re seeing protein aggregation, you may want to consider changing your incubation temperature. Some proteins...aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10–20 minutes) at 70 °C, ...If you need very accurate protein measurements, consider using an unstained protein ladder, which will ...

...Cas9 orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 orthologue... essential genes in a wide variety of bacteria including in pooled, genome-wide, formats. Interested in...dCas9 variant as well. Furthermore, they provide a high-fidelity (HF) variant of the BE3 that further improves... Then, pCRISPR is introduced along with an oligonucleotide repair template. Through recombineering, the...in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive natural products. To easily...INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) and the Zhang lab's system...select genes in E. coli. Furthermore, to demonstrate wider applicability they demonstrated success of Cas9 ...

...our CRISPR guide for a refresher on CRISPR/Cas9.  Figure 1: Cpf1 is a single RNA-guided endonuclease...crRNA/tracrRNA hybrid guides needed for Cas9 function. Since both Cpf1 and its guide RNAs are smaller than...Cpf1 functions differently. Since Cpf1 loci are widely distributed across bacterial species, Zetsche et... crRNAs for Cpf1-mediated cleavage are 42-44 nucleotides in length, about the same size as Cas9’s, but... cleaves in a staggered fashion, creating a 5 nucleotide 5’ overhang 18-23 bases away from the PAM. With...SpCas9. Since Cpf1 doesn’t require a tracrRNA, crRNA guides are only ~42 nt long. Direct synthesis of these...and off-target efficiency When Cpf1 was first identified, we didn't know much about its on-target and ...

...a march in support of science, I loved the idea and decided to go. At my local March for Science in Albany...advocate for scientist participation in government, evidence to inform policies, a protected place to work,...hired hinges on performing near miracles. When evidence does not affirm our perception of how things are...scientists are tempted to seek out alternative evidence and discard inconvenient findings. Yet, we cannot...scientists to rally in Ottawa to protest the “death of evidence,” and to speak out in unprecedented force during... and actions beyond marches, as well as allies outside the scientific community. Vanessa Sung is currently...Centre at McGill University in Montreal, Canada.  Outside the lab, she dabbles in science policy, public ...

...DNA edits and to perform functional screens to identify genes involved in a phenotype of interest. This...discuss differences between these approaches, and provide updates on how best to design gRNAs. You can also...gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment with CRISPR ...expression. Location and sequence are important considerations for designing your gRNAs. For indels, it's ...off targets. For CRISPRa and CRISPRi, these considerations are of roughly equal importance (target should...since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs to 5 – 65% of the protein ...with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development of SaCas9, NmeCas9...

...programmable guide sequence as the spacer and the remaining sequence as the scaffold. gRNA: "Guide RNA" is ...between the guide and target in order to cut DNA, whereas some mismatches can be tolerated outside this region...CRISPR array. The CRISPR array is composed of identical repeat sequences and variable spacer sequences...includes the crRNA and tracrRNA sequences in a “single-guide RNA” that performs both functions. Cas9 cuts both...all have in common is the ability to use an RNA guide to target other nucleic acids—usually DNA—to cut... sequences in the CRISPR array that will become guides for future targeted degradation. Before integration...foreign DNA that Cas proteins recognize using an RNA guide. Non-target strand: The paired strand of foreign...

...do you identify the actively silenced ones? Here, I’ll cover some screening methods to identify them. ...RNAi-trigger type you decide, any RNAi experiment benefits from an easy method to identify and quantify actively... example, while gene knockouts have long been considered the “gold-standard” for LOF studies, the high...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation...the importance of using orthogonal methods to confidently conclude the function of a gene of interest. ...RNAi activation. A typical RNAi experiment can be divided into three fundamental steps: creation of the ...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based...

...with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and...gene delivery capsids previously identified in rodents have not been widely successful in primates. The authors...articles.  Here's what you'll find in this post: Peptide-assisted genome editing for primary cells Monomeric...delivery Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for primary cells By Susanna...and co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining Cas9-PAGE with...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused...reporter (mChe), with co-incubation with assist peptide (AP). C) Workflow for Cas-RNP-PAGE editing of primary...

...putting these into slide format. You want people to focus on you and your ideas. Slides will be     distracting...also be a slide deck, markers, etc. Chalk or markers force people to be more succinct. Slide decks leads...is vague. Instead, consider "A mechanistic understanding of this system will provide the first defined ...avoid the temptation to provide too many details. Reverse engineer your talk. Identify the key points you ...original blog post below! --Ed.  Eight years ago, I decided to write a "how to" manual on applying for faculty...the trials and tribulations of the process, I’ve provided guidance and mentoring to several people that ...failure to clearly articulate one's talents and ideas. Some key points from my ebook, "Applying for a ...

...programs provide support for academic career development, it's often left to students to identify and build... design variables need consideration? Enrich your knowledge and skills outside of the lab Effective verbal...you build a network outside of the academic world Participating in activities outside of the lab expands...and government. This great resource allows the undecided to learn about a variety of science professions...on their skills and interests. Once scientists identify potential career pathways, education and mastering...mastering new skill sets must occur by doing work outside of the lab. Extracurricular activities don't have...tool and technique development. However, careers outside of the lab highlight “transferable” skills such...

...specificity is dictated by a DNA guide sequence rather than an RNA guide as with Cas9. Third, it can be ...with an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo...Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids. Cas9 guide RNA delivery does however...sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo is ...have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’-phosphorylated in house...modified from a company. However, unlike an RNA guide, it cannot be produced from a plasmid in cells. ...Try NgAgo for Yourself On the other hand, the widespread success of CRISPR-Cas9 across multiple model ...

...Yiqun, et al. "CRISPR Cas9-guided chromatin immunoprecipitation identifies miR483 as an epigenetic modulator...system, consisting of dCas9 and targeting gRNA, to identify molecular interactions at this site (Fujita and...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...different fields of study. In addition to the identification of DNA binding proteins, this method can characterize...sensitive and specific and allowed the Xu lab to identify protein and nucleic acid binding partners at numerous...various biological fields. In 2015, the Fujii Lab identified non-coding RNAs associated with telomeres by ... malignancies. Additionally, the Tapscott Lab identified regulators of DUX4, a transcriptional regulator...

...skills guide Resources on Addgene.org Learn more about Careers at Addgene Watch out career videos  ...training. Similarly, there are several factors to consider when selecting a postdoc mentor and lab. These...Depending on the position, you may also want some evidence of teaching experience. How do you get there... having sufficient data to write a manuscript. Consider questions like: What figures are missing? For ...explore your system more, you will have a better idea of how to leverage it to ask new and interesting... grad school, you searched for and eventually identified a postdoctoral mentor. A good mentor typically...typically writes the grants that fund your research, guides the research directions of the lab, gives you constructive...

...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...

...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...

...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...

...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...

...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...

...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...