...enough to be expressed in bacterial and other cell systems. Since the majority of these alternative reagents...Antibody GuideRecombinant antibodies available from AddgeneBrowse our recombinant antibody plasmid collection...I love Addgene's new Antibody Guide and not just because I helped create it. But while antibodies, as...could take the needed steps to continue where the guide left off - namely, a blog post devoted to the other... of disulfide bonds, monobodies can be easily expressed in transfected eukaryotic cells, allowing for ...Aptamers are not proteins at all. These high-affinity RNA molecules can bind to a wide variety of targets, ...Resources Additional resources on the Addgene blog Plasmids 101: Aptamer FluorophoresAntibodies 101: scFVs...

...ligand-bound GPCRs of interest. The TRUPATH plasmid kit consists of 20 plasmids encoding 14 alpha, 2 beta, and 4 ...inhibition). AAV preps from DREADD plasmids allow you to easily express a DREADD receptor in a specific ...quick guide to chemogenetics Resources on Addgene.org Check out Addgene's chemogenetics guide Find chemogenetics...response? Let’s take a look at a few examples with plasmids deposited at Addgene.  PRESTO-TANGO: The ligands...resource called PRESTO-TANGO (parallel receptorome expression and screening via transcriptional output - TANGO...factor from the receptor, which can then activate expression of a luciferase reporter in the nucleus. The ...Bryan Roth’s lab also assembled a collection of plasmids to elucidate the role of 14 of the 16 non-visual...

...Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...an extensive RNA scaffold and a domain needed for DNA unwinding. They’ve deposited plasmids for active ...for E. coli and human expression. Read the paper in Nature Find the CasX plasmids Drug inducible transcriptional...vector sequence. Homology arms that flank the donor plasmid cargo are complementary to either side of the double-stranded... Acids Research Find the drug inducible Cas9 plasmids E. coli genome-wide inhibition library In the...targeting requires two sgRNAS: one that targets the knock-in site and a universal sgRNA that targets the donor...

...consisted of three plasmids: the packaging, envelope, and transfer plasmids. The packaging plasmid carried a mutated... packaging and envelope plasmids are two of Addgene’s most requested plasmids! Lentiviral particles with... make stable transgene-expressing or knockout cell lines. Many CRISPR plasmids are designed for lentiviral... of Addgene’s most requested plasmids are used with lentiviral expression systems. If you’re new to the...lentivirus, check out our lentiviral plasmid and biosafety guides before beginning your experiment. Let...into the genome, permitting long-term transgene expression, but they could only infect actively dividing...non-dividing cells, but without sustained transgene expression. To design a viral vector system that could do...

...domain. These mutations result in an inactive RNA-guided DNA binding protein (dCas9). dCas9 is then tethered...et al., 2014). The sgRNA or tracrRNA sequences can also be modified to include RNA domains that recruit...Sanson et al., 2018) . This tracrRNA was combined with the sgRNA expression vector allowing the screen ...CRISPRa) to regulate gene expression. Some CRISPR libraries contain many sgRNAs per gene to increase confidence...Calabrese: human CRISPRa sgRNA library In addition to inhibiting gene expression, dCas9 can also be used... that consists of inactive Cas9-VP64, a sgRNA with two MS2 RNA aptamers, and the  MS2-P65-HSF1 helper ...screens can help reveal the function of lowly expressed genes or genes that are more drastically affected...

...recombinant adenoviral plasmid construct, please see Addgene’s Adenoviral Guide webpage, and references...Resources on Addgene.org Find Adenovirus Plasmids Adenovirus Guide Page  ...death of transduced cells and loss of transgene expression (Interestingly, the strong immunogenicity of ...capacity is limited to 8.2 kb, and in vivo transgene expression ceases relatively quickly due to immune response... capacity (up to 36 kb!), long-term transgene expression and negligible toxicity. However, their production...down into 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular...adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene of interest...

...resources from Addgene Plasmids 101: Modular Cloning Addgene's CRISPR Guide Plasmids 101: Golden Gate Cloning...restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just ...correctly banded clones for plasmid sequencing and found 98% of the plasmids were perfectly correct. Because...pRDA_757 (Piggybac; constitutive expression) Mammalian Plasmid pRDA_791 (contains only orio and...the assembled plasmid. It shows you each component, with the ID of the component plasmid, component name...the component plasmids on the Addgene website. Step 4: Naming your assembled plasmid and generating...structure takes a lot of the design work out of the plasmid creation process. Each module of Fragmid represents...

...more on this below) it can be incubated or co-expressed with avidin or a structurally similar protein ...ABC and IHC check out Thermo Fisher Scientific's guide. There are an array of other molecular biology applications... been shown to effectively biotinylate AviTag expressing proteins in bacteria, yeast, insect and mammalian...biotinylating proteins with BirA. You can find plasmids that contain both AviTag and BirA at Addgene! ...Photobiotin can be used to non-selectively label DNA and RNA. Photobiotin acetate consists of a biotin molecule...., & Zhu, Y. “A non-radioactive method for small RNA detection by northern blotting.” Rice 7.1 (2014):... shift assay to examine specific dsDNA, ssDNA or RNA–protein interactions.” Nucleic Acids Research, 23.18...

... binds the guide RNA (shown as mature crRNA) and binds the RNA target before initiating RNA cleavage. ...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with... exclusively targets RNA. Cas13 has become a natural successor to traditional RNA interference, as it ...target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing. There are four...Additional resources on Addgene.org CRISPR guide CRISPR plasmids CRISPR references ... you need to ensure every unit of the complex expresses at similar levels. It was only recently that researchers... researchers began building optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems...

... Visit Our Stem Cell Page Find Plasmids for Reprogramming Find Plasmids for Differentiation ...reprogramming differentiated adult cells by the ectopic expression of four embryonic genes- OCT4, SOX2, KLF4, and...to a Nobel Prize for Dr. Yamanaka in 2012. Find plasmids for generating iPSCs iPSCs and ESCs closely resemble...possess similar morphologies, growth properties, and express stem cell markers similar to ESCs [11].  While ...Pharmacological Sciences, a Cell Press reviews journal. Stem cells are special types of cells that can...Pharmacological Sciences, a Cell Press reviews journal. She joined Cell Press to pursue a career in science...

... one, Ly6a, showed differential protein and RNA expression between the brains of C57BL/6J mice and BALB...Deverman Lab when they looked at LY6A protein and RNA expression patterns, the PHP.B permissive phenotype of...phenotypic effects such as those that affected gene expression, mRNA splicing, or protein coding regions. In total...strong linkage to the PHP.B phenotype and its expression pattern in the mouse brain.  Both labs then took...Deverman lab started by seeing how changes in LY6A’s expression affected PHP.B’s transduction rates. Knock out...still transduced by PHP.B but only if Ly6a was expressed. Additionally when AAVR knockout mice were IV ...permissive and non-permissive mouse strains. PHP.B plasmids and PHP.eB viral preps are available from Addgene...

...high expression, so adding this G could help with expression, though it is possible for the plasmid to ...use double nickase, you could express two spacers and use PX335 to express the Cas9n (nickase). The concept...double nickase system is that you can express two different chimeric gRNAs with the Cas9 nickase which will... frequently requested CRISPR plasmids at Addgene Find protocols and gRNA design tools: List of CRISPR...optimized for specific plasmids Links to different software to help you identify your gRNA target sequences ...articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions...genome editing using the Zhang lab's CRISPR/Cas plasmids. Designing your CRISPR genome editing experiment...

... multiple plasmids. Second generation packaging systems use 3 plasmids; an envelope plasmid that usually...encodes VSV-G, a packaging plasmid containing structural genes and a transfer plasmid carrying the transfer...Rev gene from the packaging plasmid and incorporating it into a fourth plasmid. In addition, the promoter...and packaging plasmids in order to obtain the best titers. Requiring an additional plasmid for transfection... titer virus for transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest...Resources on Addgene.org Lentiviral Guide Find lentivirus plasmids Browse all viral vectors ... a usable titer, or fine-tuning selection and expression in your target cell line, researchers often find...

...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no proteins...involved in RNA based surveillance of repetitive gene families and provide a way to guide inheritance ...finding in the regulatory RNA field. The Rajewsky lab is studying circular RNAs (who knew…I had barely heard...stranded, covalently closed RNAs seem to be conserved in mammals, expressed in neurons, and are especially...against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron...understanding complex regulation of and by tRNAs and tRNA-derived RNAs. Stay tuned for big developments in this... discover circRNAs in RNA-seq data. As far as I can tell, we don’t really know what circRNAs do yet or...

...Cre-based Brainbow-expressing cells. Autobow plasmids are all-in-one versions which express self-excising ... host cells in order to optimize expression. The expression plasmids for a given species are based on ... Hot Plasmid list. Other popular plasmids & blog posts @Addgene: The 10 Most Distributed Plasmid Technologies...inducible inactivation. Find the Robinson plasmids or the Royle plasmids and get to work! Robinson et al...descriptions of various optogenetics plasmid tools, visit Addgene’s optogenetics guide. More optogenetics tools...designed the pOSIP (one-step integration plasmid) series of plasmids, vectors that convey both the sequence...Chen, pCS2-3xFLAG-NLS-SpCas9-NLS is a Cas9 expression plasmid that was used by Chen and colleagues for ...

...lentiCRISPRv2, expressing both Cas9 and an individual gRNA from this single vector) and guide-only (lentiGuide...gRNA plasmids targeting the human kinome. These plasmids contain the same 3-4 gRNAs found in the first ...setting. The individual plasmids will also be useful for imaging or gene expression assays necessitating ...lentiGuide expressing only an gRNA, for use with cells that already have Cas9). Each library consists of ...individual plasmid formats A first for a library at Addgene, you can also order the individual lentiGuide gRNA...individual plasmids separately. Additionally, this set can be used for cherry-picking individual gRNAs that ...Pooled Library Page Find Validated gRNAs Deposit Your CRISPR Plasmids ...

...with the guide RNA.   Non-target strand The DNA strand that does not base pair with the guide RNA. Despite...Acronym Full name Notes CRISPR terms gRNA/sgRNA Guide RNA RNA used by Cas9 to locate a matching DNA ...structure. In order to bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent... called an R-loop (Figure 2). This allows the guide RNA to base pair with the DNA target strand, leaving...binding to DNA and separating the two strands. The guide RNA base pairs to the target strand. The complementary...Addgene.org Browse CRISPR plasmids for base editing Get a broad overview with our CRISPR Guide Check out our CRISPR... reduce its tendency to make off-target edits to RNA. Like other ABEs, the editor they were using consisted...

...three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid. These...pseudotyped protein is encoded on the envelope plasmid which contains a promoter, the envelope gene, and...virus, HIV-2, and human foamy virus in cell lines expressing galactosyl(alpha1-3)galactosyl (alphaGal) sugars...than viruses produced in cell lines that do not express these sugars. (Takeuchi et al., 1997). The alphaGal...scientists encode the spike protein on the envelope plasmid. Image from Crawford et al., 2020.   Broad tropism...infecting most cells, but not all. For example, LDLR expression in hematopoietic stem cells, T cells, and B cells...pseudotyped virus requires the upregulation of LDLR expression using other methods.  In terms of preparation...

...Check out our Optogenetics Guide or browse our collections of optogenetics plasmids – and keep an eye out ...resources on Addgene.org Optogenetics Guide Optogenetics Plasmids References Aljiboury, A. A., Ingram, ...clusters under blue light Check out our Optogenetics Guide to learn more about how these modules work, and ... of katanin to that anchor. So, you’ll need to express both the EB3N-VVDfast-iLID anchor module and the...module, which are also available as lentiviral plasmids if you want to make a stable cell line. This design...specific area at the plasma membrane (Figure 2). Just express the plasma membrane-localized iLID along with your...Opto-RhoGEF design and activity. B) OptoTIAM (magenta) expressed in a monolayer of endothelial cells, showing cell...

...DNA or RNA purification to avoid contamination, take extra care when performing RNA extraction. RNA is inherently...cover the basics of isolating RNA without a kit. Tips for working with RNA (whether you are using a kit... freezer to prevent RNA degradation. They will degrade in a -20°C freezer.   RNA extraction methods evolved...activity upon cell lysis, RNA is degraded. To effectively isolate intact RNA, a fast, strong protein denaturant...separate the intact RNA from DNA and proteins. Although very effective at isolating total RNA, this method requires...and RNA can then be isopropanol precipitated. This method reduced the time it took to isolate RNA from...precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but not DNA and...