...even CRISPR based gene editing. But, how do you decide which tools to use, and what do you need to know...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...signaling pathways. Light exposure removes the block provided by the LOV domain allowing the pathway to be activated...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...activate or inhibit a cell in succession and maintain fidelity with the stimulus. For example, if you want to...

... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B). To label...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...interpret. Check out (Jambor et al., 2021) for more guidelines for creating clear and informative figures. Pro-tip...

...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...downstream of the promoter found in the backbone. Ideally, the backbone will contain a variety of restriction...plasmid backbone looks like the one found on the left side of the image below. It has a promoter (blue arrow...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...Methylene Blue Post Visible Light 100 Ethidium Bromide Pre and Post UV Light 0.5 For more information...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...about the company and what the goals were and so I decided to join. I’ve been working with Addgene since 2014...distribution of the plasmids and their ability to identify and fulfill a need in terms of helping the scientific...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...perspective, what do you think are important considerations for the future of open science? What about ...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

...2004, that was a fairly simple goal… and we had no idea how much our understanding of what accessibility...did! From Addgene's very beginning, we had to consider digital accessibility for our website and online... spaces. Alt text is used by screen readers to provide visually impaired visitors with information about...educational content conflicted with plain language guidelines.  Which is why, at the Society for Neuroscience...our workshop how they felt when they read papers outside their field, the answers were primarily negative... Ultracentrifugation First, you’ll want to identify the reader’s goal. Why are they reading this page...and learning how to purify AAV.  Second, you’ll identify your purpose in using that image. Try using the...

...cloning strategy? A screen will help you more easily identify successful clones so you have to weed through ...relies on antibiotic resistance. But what about identifying clones that contain your desired insert? This...ampicillin plates containing X-gal allows you to identify cells that took up the vector and distinguish ...at a few screening methods. Blue-white screen A widely used method of screening is the blue-white screen...example, choose enzymes that only cut once on either side of the expected insert. If the plasmid contains ...planning to go this route, check out our protocol video on using restriction digests to analyze plasmids...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...

...engineering designs and ideas ranged from a biological breathalyzer to pesticide breakdown, production ...engineering of biology. Participating schools are provided with synthetic biology lab kits and lesson plans...recognized name in the field of STEM education worldwide. The BioBuilder curriculum is now taught in almost...and teachers can investigate together. Teachers decide which BioBuilder lessons to teach, and how — some...know-how to design/build/test their own project ideas using synthetic biology.Launching with a kickoff...newsletters, pairs teams with STEM mentors, and provides a nationally recognized platform in March to exhibit...Synthetic Biology Blog Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids for Creating...

...binds to the target DNA sequence as directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats...c-di-GMP in response to far-red light (730 nm). This nucleotide triggers the translocation of the protein BldD...Simply expressing anti-CRISPR proteins does not provide much control over when Cas9 activity is blocked...2020). Fluorescent dCas9 proteins can be used to identify specific genomic loci for imaging techniques. ...architecture. Yu et al., 2020 FAST BphS from R. sphaeroides Split Cas9 Genome editing by NHEJ and HDR. Can...Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018 ...al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...

...protocols Find protocol videos on our website Read our Molecular Biology Guide ...material. Then, the homogenate was loaded onto cesium-chloride gradients and ultracentrifuged for up to 20 hours...However, instead of RNA separation using cesium-chloride gradients, water-saturated phenol, sodium acetate... and variations on this no-kit method are still widely used today (Chomcynski and Sacchi, 2006).  View... tissue collection and homogenization. The most widely known of these products is TRIzol® (also called...Allewell and Sarma, 1974). Additionally, ThermoFisher provides a protocol on how to integrate RNAlater® with ... trick is to precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but...

...single nucleotide polymorphisms. Fast facts – PAM requirement: none. Best for: high fidelity ssDNA targeting...engineering powerhouse, and for good reason. It is guided by an RNA molecule, the gRNA, to a complementary... and an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What ...ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a single mismatch in the target...won’t cut it – literally. Cas13 Cas13 is an RNA-guided nuclease, however, unlike Cas9, it targets RNA ...Abudayyeh, O., Omar, et. al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell...Website CRISPR Plasmids and Resources CRISPR Guide  ...

.... "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library... will introduce you to this new technology, and guide you through the basics of CombiGEM-CRISPR experiments...with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome editing in large-scale has... has led to the rapid generation of genome-wide gRNA libraries that knock out (Doench et al., 2016; Hart...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that.... Ma, Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell...Catch up on Your CRISPR Background with Our CRISPR Guide Pages Find CRISPR Plasmids Find More Tools for Combinatorial...

...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method...traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base...such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result, these sites ...exploits the ability of Type IIS enzymes to cut outside their recognition site and permits DNA fragments...Gate cloning for inserting the appropriate oligonucleotides specifying a gRNA target sequence into a Cas9...amenable to "domestication", you might want to consider using an alternative method like Gateway cloning...cloning or Gibson assembly. Another important consideration is the design of flanking overhangs. Although...

...to use them, what they can tell you, and how to identify which one is right for you. Statistics – The ...permutation of pairs within the five is different. To identify significant pairings, additional tests, referred...hoc test, done after your initial analysis can provide information on specifically which groups are statistically...within your population? Sometimes this can’t be avoided, but if it can be, you can reduce the family-wise... p value less than or equal to the alpha value divided by the number of null hypotheses. Benjamini uses...uses a complementary approach which identifies a p value cut off. This method entails arranging all calculated... by finding the largest p value where its rank divided by the total number of comparisons performed multiplied...

...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com. How ...al., 2016). Target-AID acts similarly but not identically to BE3, modifying a 3-5 base window 18 bases ...editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with... created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3...positions 4-7. ABEs 6.3, 7.8, and 7.9 display slightly wider editing windows of position 4-9, although editing...Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their other experiments...

...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity... fast-acting optogenetic tools. The advantages provided by chemogenetics are instead that it is noninvasive... crossing the blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies...response you aim to manipulate. It is important to consider that the same receptor can elicit different responses...muscarinic receptor derived DREADDs has been clozapine-N-oxide (CNO). However, concerns have been raised over the...Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...

...isotype choice. Target and application should be considered The first question you may have is whether a ...monoclonal primary antibody (A). To boost signal, consider using subclass specific secondary antibodies instead...strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal antibody you may wonder, “Will ...immunoprecipitations. Your sample species should also be considered when choosing an antibody. In some applications...options are available to you, it may be worth considering an antibody raised in a different species.    ...      Table 1: Isotype considerations when choosing antibodies   Complexity is a key factor...with a distinctly conjugated secondary antibody. Consider too that some secondary antibodies can cross react...

...You’ll want to set up your blot with a polyvinylidene difluoride (PVDF) membrane, which is sturdier and...Western blots are a great tool to identify a protein of interest in a complicated solution like cell ...already run your blot using nitrocellulose, you may decide you have nothing to lose by trying to strip and...abundance of your targets isn’t too different, consider antibody affinity: stronger binding antibodies...matter what stripping methods you throw at it. Ideally, dry your membrane once before any detection steps...(βME) 100–115 mM = 0.7%–0.8% (v/v) Breaks disulfide bonds HCl Adjust pH to 6.7–6.8 Adjust ...addgene.org Western Blot Protocol Western Blot Protocol Video ...

... in genetically identical mice. Thus, scientists must genotype each animal to identify the ones with the...-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...traits and genetically engineered mouse models provide researchers with useful and adaptable tools to ...background, which means that each mouse is genetically identical and all mice of the strain are homozygous at nearly...inserted gene may have errors introduced to the nucleotide sequence during the DNA repair process. The addition...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...

...sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences or annotated...to check the quality of other services that we provide, including viral preps and pooled libraries. As...this changeover had no impact on our ability to provide requesting scientists high quality data! Addgene's...culture to prepare it for sequencing, we feel confident that these few minor mismatches usually don’t ...differs from what we would expect given the data provided by the depositing lab, we call these “quality ...Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations...

... 2021,  with over 70,000 articles in PubMed, the idea that DNA methylation is linked to disease has now...tools to specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started...Recently, however, a particular tool has gained widespread popularity – and notoriety – as a tool of choice...   Let me leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest...interest. Having the CpG directly inside the gRNA sequence is a great guarantee that DNMT1 will be blocked...Cas9 Resources at Addgene.org Read our CRISPR Guide to Epigenetics References Charlton J, Jung EJ, ...Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. ...