We narrowed to 718 results for: des.1

...centrifugation (lenti) Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher...will help you decide which is right for you. Table 1: Mammalian versus insect cells for viral production... (about three to four days), this second round provides the added benefit of producing a stock P1 virus...

... fusions that retain their individual activities (1). Researchers also discovered the modular format of...systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown ...ubiquitin system has been devised (8). References 1. Casadaban M.J., Martinez-Arias A., Shapira S.K., ...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ...

...and is recommended for use in western blots (Figure 1). If you prefer to produce your own antibody or want...are also available from Addgene!   Figure 1: Experimental validation for Anti-V5 [IPI-SV5-Pk1]...biophysical studies of protein degraders. Nat Commun., 15(1), 8885. https://doi.org/10.1038/s41467-024-52871-9...promising tool for future structure-guided drug design and studies of protein ubiquitination and degradation...CRBNmidi plasmid here!   Kroupova, A. et al. (2024). Design of a Cereblon construct for crystallographic and...specifically for C. elegans (Cao et al., 2024). The team designed coding sequences for mScarlet3 and mScarlet-I3...

...animal will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models... can be explained primarily in two different ways 1) your hypothesis is wrong or 2) there is some problem...follow him on Twitter @derekpsimonphd.    References 1. Stuber, Garret D., et al. "Excitatory transmission...other words, your optogenetic experiment should be designed around a behavioral test in which your circuit...of mouse transgenics. Everything that has been described is intended for the mouse; however, all the steps....Diester, Ilka, et al. "An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011)...

...the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started off...form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which, in...standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments....daydreaming about sunny days in the UK.   References 1. Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis...knock-in. Another good design tool can be found at Desktop Genetics .For ssODN design, remember to incorporate... systems, you will have to design your crRNAs using other web-based design tools (for C-terminus tagging... not work in pull-down assays: Epitope tagging provides a solution Proteins - the workhorses of the cell...

... this blog post. I separate sgRNA design tools into database and de novo solutions. Database tools allow...automated rational design. The first of only three such databases, EENdb (published 1 Jan 2013, no longer...community in order to succeed. With respect to de novo sgRNA design, I again separate it into two categories...follow him on Twitter @CMacPhD.          References  1. Ghorbal, Mehdi, et al. "Genome editing in the human...single-target design”, “multi-target design”, “off-target aware”, “high mismatch limit”, “approximate design”, “... “empirical design”, “single-PAM design”, and “multi-PAM design”. Trends: By the end of 2014, tools began...with the post-design process such as primer and plasmid design. Examples:  “multiplex design”, “multi-method...

...sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson et...):1028-34. doi: 10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed. Find plasmids from this publication at ... the summer of 2013, a remarkable nine papers describing CRISPR/Cas9 genome engineering methods for C....America. To use this SEC-based system, first design and clone 500-700 bp homology arms for your gene...injected 60-80 animals per gene, they obtained the desired insertions on the first attempt for 6/7 genes. ...

... RNAs inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated ...actually does, and neither plasmid is maintained (Figure 1).  The partitioning problem Plasmid incompatibility...Agrobacterium tumefaciens. Genes & Genetic Systems 77:1–9 . https://doi.org/10.1266/ggs.77.1 Pubmed PMID: ... Environmental Plasmids blog). Scientists have designed small, high copy incompatible plasmids that lead...Pubmed PMID: 25889267 Ebersbach G, Sherratt DJ, Gerdes K (2005) Partition-associated incompatibility caused...

...Check out Addgene's New Viral Service! References 1. Hay, R.J., Macy, M.L. & Chen, T.R. “Mycoplasma infection...contaminations.” Methods in Molecular Biology 946 (2013). 1-13. Pubmed PMID: 23179822. 5. Segura, M.M., Kamen,...Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors."Nature protocols 4.4 (2009...best to start from scratch. Obtain a vial of the desired cell line from a reputable distributor such as ...extreme changes in osmotic pressure and pH can destabilize the viral envelope. One drawback of the multiple...

...nLightR (Kagiampaki et al., 2023).     Figure 1: Properties of nLightG and nLightR. A, Structural ...Article 6. https://doi.org/10.1038/s41592-022-01488-1 More resources on the Addgene blog Fluorescent proteins...will need to be accounted for in your experimental design. Find nLight plasmids here! Next steps While nLightG...

...Rodríguez et al., 2011).  Genetics of SPARC Figure 1: SPARC and SPARC2 use the GAL4-UAS system along with... several common genetic tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site....References: del Valle Rodríguez A, Didiano D, Desplan C (2011) Power tools for gene expression and clonal...

...refreshed figures made using BioRender.com. Figure 1: PRIME editing Please check out the updated guide...update, we have added new sections to share and describe some incredible new CRISPR technologies developed...much-anticipated prime editing section and additions describing CRISPR’s ability to make large genome edits using...transposases or the Cascade-Cas3 system. We also describe newly engineered Cas9s with more flexible requirements...

...following three strategies might help!     Figure 1: Species on species staining can be a problem because...same host species as your secondary antibody since 1) your secondary should not recognize Fab fragments...immunocytochemistry (ICC). Not all of the solutions described here may be as effective for other techniques,...

...and take a look at each of its parts. Figure 1: Parts of an AAV transfer plasmid. Parts shown in ..., ubiquitous expression, while the human synapsin 1 promoter is neuronal specific (Haery et al., 2019)...this post, you’ve got the knowledge to be able to design a construct for the successful and specific expression...

...from distantly related organisms. In 2006, less than 1% of the genes deposited at Addgene were synthetic....synthetic biology is expanding rapidly.  References 1. Kunjapur, Aditya M., Philipp Pfingstag, and Neil ...of 173 known synthetic and natural genes.  In designing and optimizing their algorithm, they found that...

...defined as (1) the struggle between two opposing forces or (2) the result of competing desires or (3) the...journey even when they know the ending.    Fig. 1: We all know that the superheroes win in the end. ...

...(the Gal4/UAS system), and the third post will describe how you can make your own mutant flies. Find Drosophila...

...the journal-specific guidelines.  Peer-review step 1: The first read At the first read of the paper, I ...group of Prof. Mark Tester, where she focuses on (1) salt-induced changes in root-to-shoot ratio in Arabidopsis...of my review - "The manuscript by ZZZ et al., describes ... The authors share a wealth of data on ... ...Additionally, you can ask that data and resources described in the manuscript are accessible to the community...

...blotting, named for its inventor Edward M. Southern (1), can be used to detect DNA identity, size, and abundance...hours at low cost with restriction mapping (Figure 1). Though some labs may prefer to send DNA directly... serotonergic system and behavior.     References 1. Southern, Edwin Mellor. "Detection of specific sequences...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands of all...

...native endonuclease activity degrades the displaced strand.   Figure 1: EvolvR consists of an error-prone...mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR,...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...turbidostat. Only mutate particular types of nucleotides. Both the Bassik Lab and the Chang Lab have created...

...overcomes many of those limitations (Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule...GTPase Signaling Toolbox. ACS Synthetic Biology, 11(1), 515–521. https://doi.org/10.1021/acssynbio.1c00604...optogenetics than neuronal activity. Researchers have designed a variety of photoswitchable elements into many... and limitations – for example, microtubule-destabilizing drugs are impossible to target to specific subcellular...plasmids if you want to make a stable cell line. This design allows highly localized, rapid and reversible microtubule...Rho GTPase activity. A) Schematic of Opto-RhoGEF design and activity. B) OptoTIAM (magenta) expressed in...processes in cells and tissues. And, this Opto-RhoGEF design could be extended to other GTPases. The Chow Lab...

...sticks out and can be considered a linker (figure 1). In fact, for several FRET biosensors (yellow cameleon...fluorescent protein are relatively close together (figure 1). Where should the fluorescent protein be inserted...fluorescent protein database." Nature methods (2019): 1. PubMed PMID: 30886412. Mastop, Marieke, et al. "A...any DNA sequence in the way that they like, the design and engineering of fusion proteins is relatively...fusion proteins and highlight some aspects of their design.  Protein size and shape matters The green fluorescent...necessary for post-translational modifications. Despite these potential issues, GFP has been used successfully...consist of random amino acid sequences or (ii) designed to form an inert, unstructured peptide and therefore...

...may find “Chapter 1: What is an Antibody” quite useful. If you’re looking to design your first flow cytometry...eBooks, the Antibodies 101 eBook is designed to be a helpful desktop reference. Each chapter brings together...

...by Jason Nasse.  References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant permits efficient... There are two methods of neuronal tracing (Fig. 1): one to visualize neurons that send information to...trillion connections (synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it... this can cause challenges for the experimental design as these vectors can exhibit spurious expression...efficiency can have a major impact on experimental design and interpretation of results. Take extra care ...

...speed: base editing before Cas lets go Figure 1: David Liu gives a talk at the Cell Symposia: Gene..., Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution...

...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be used as a model to relate these...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...pathological developments (Vieira et al., 2019) Besides their ability to regenerate, the axolotl has several...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...Arabidopsis genome project marked the beginning of widespread Arabidopsis genetics research development. Arabidopsis...allows for easy cloning of constructs specifically designed for use in Arabidopsis. There are also important...

...distinguish between your two targets.    Figure 1: In the isotype conversion process the variable regions...natural isotype to that of a different group (Fig. 1). This process, called isotope conversion, gives users...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...

...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...quantified first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary...store your plasmid DNA Learn about the different grades of plasmid DNA preps Resources on Addgene.org ...

...agarose gel. The resulting gel image includes a 1kb ladder (lane 1) that has bands ranging from about 500bp...restriction digests are comprised of 2 separate steps: 1) incubating your DNA with restriction enzymes which...

...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements...induction of a promoter. Below, you’ll find a short description of these three types of inducible promoters, ... popular light-regulated two component systems designed by the Tabor lab. Which inducible promoter is ...

... https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing by: Rachel... Zebrafish) (Xiong, et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system... error rate for long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving ...nanobody-GFP interaction). The modularity of the system provides researchers better flexibility and makes the approach...other experiments. And, while the BLITZ system was designed and optimized with zebrafish in mind, it can easily...Recently, Ravid Straussman’s lab has developed visual barcodes for clonal multiplexing of cells in live imaging...peptide. By combining three different localization peptides with four different fluorescent proteins, and ...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova...have pre-designed plasmids for these systems! Ion sensors One common biosensor class is designed to monitor...all have specific sensors designed for them. For example, this class includes tension monitors associated...interest for many disciplines! Most of the sensors designed for calcium generate a fluorescent output, which... fluorescence in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium.... Physiological factors Unlike the previously described sensors, this class measures either factors as...to use them, you’ll need to use the vectors (or redesign the tools). It’s a good thing, though! Viral delivery...

...correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...complementary to standard sequencing primers. If not, design primers to sequence your insert. When you get your...

...central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand ...fluoresce: FlipCherry. FlipCherry provides a proof of concept that the designed used in FlipGFP can be applied...range and high brightness. The Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which ...prevent binding, the β strands in FlipGFP were redesigned so that they are parallel to each other and thus...fluorogenic reporter toolkit, the Shu Lab also designed a red fluorogenic protease reporter based on superfolder...PMCID: PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for...Central PMCID: PMC5026494. Zhang, Qiang, et al. "Designing a green fluorogenic protease reporter by flipping...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element...infectivity): The use of the VSV-G envelope provides the widest tropism or range of cells a virus can infect...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...lentiviral packaging methods. The three components described above (envelope, packaging, and transfer) are ...into the 293T packaging cell line. This system provides the greatest flexibility to pseudotype γ-retrovirus...nomenclature of the plasmid backbone is meant to be descriptive of the components found within the backbone. ...

...'ll want to design a custom primer. Because Sanger sequencing can usually sequence just 1 kb of DNA, custom...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...is not enough on its own to help you find your desired plasmid. In many cases, you will need a screening...you will need to sort through them to find the desired clone. Do your selection, then screen Why add a...what about identifying clones that contain your desired insert? This is where a  screen comes in. Let’s...blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...

...versions with toxicity to many organisms. Fig. 1: Exogenous sources of dsRNA Cellular sources of... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...into agriculture as species-specific pesticides and insecticides, an advancement over previous chemical...prone to degradation – those are the words that describe RNA! Double stranded? Well, that’s just for DNA...also known as RNAi, are short, dsRNA molecules designed to degrade mRNA transcripts. RNAi activates the...sequence is found (typically RNA that the RNAi is designed against), RISC can repress translation and/or ... and repetitive sequence. All of the R-loops described above play distinct roles in promoting cellular...

...This project: 1) develops state-of-the-art methods of sex and gender analysis and 2) provides case studies...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...approaches to gender equality over the past several decades: “Fix the Numbers of Women” focuses on increasing...using both sexes in basic research is excellent design), but they used all female stem cells—this was ...environmental factors. How to include sex in experimental design Recognizing the importance of analyzing sex, the... above, another excellent resource for research design is Kalpit Shah et al., “Do you know the sex of ...Corporations need to integrate these insights into product design. But eyes have been opened—and we cannot return...

... of two outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal dynamics...monitored over time by sequencing the barcodes in the population (all barcodes can be amplified using the same...with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target cells' genomes...ClonTracer system, lentiviruses containing these barcodes infect the target cell population at low multiplicity...replicate populations, they found that similar barcodes consistently made up the majority of the resistant...resistant populations. In fact, 88% of the barcodes within the resistant populations were shared by at least...

...new data). For general transfection, you could use 1 µl of 10 µM oligo stock for each well on 24-well plate...A10: The most we've tried to insert so far has been 1 kb. We used homology arms that were 800 bp long. Q11...able to cut). Clones without insertion will show a ~1 kb and ~7.5 kb fragment (both BbsI and AgeI will be...Mammalian Cells. Molecular and Cellular Biology, 18(1), 93–101. https://doi.org/10.1128/mcb.18.1.93  Ran...template vector design guidelines? A7: A few notes below are considerations for designing HR donor. Generally...usually pick 3–6 guides around the region to find the most efficient guide (most guides would work, but... this ‘split RNA’ design is not as efficient in genome editing as the second design (in pX330/PX335), ...

...addition of nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either a 1-7 base poly...poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second tRNA in the tRNA-gRNA-tRNA constructs...advantage over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors...the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely be adaptable...worries, I've summarized the three strategies described in this post in the graphic below - including ...

...plasmid, based on the assumptions outlined in Table 1.   Of course, this is for the simple insertion of...any restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...introduced, they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...more about them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...   Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral Tet-on ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

... closely data points fall to each other (see Fig. 1). Alpha values – numerical risk allowance in a ...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...values from smallest to largest and ranking them (1 being the smallest value). It calculates the cut off... been described in this blog. If your data does not meet the assumptions for the tests described above...experimental groups, but how do you determine if they deserve that oh-so coveted asterisk of significance? You...referred to as two-sample or independent, but these designations all refer to the same test. On some occasions...Correction and the Benjamini-Hochberg procedure are designed to reduce false positive rates and control for...

...Tools Atlas (Link opens in a new window) . Figure 1: Selection, prep, and testing of AAV enhancers in ...populations using systemic AAV delivery. The collection includes the top vectors with the highest specificity and...

... and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts...An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent protein...tables with fluorescent protein properties and provides a quick way to compare fluorescent proteins, e.g...been used in yeast by Lee et al (2013) and in nematodes by El Mouridi et al (2017) and Heppert et al (...practical brightness. Therefore, practical brightness provides a better picture of what can be expected in a ...

...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently...and does not consider the missing or damaged nucleotides that are common to biological sources of DSBs...better substrates for ligation. Although we do not describe these steps here, the processing of DNA ends tends...of the canonical Cas9 approach. A pair of CRISPR guides that flank regions of hundreds of base pairs or...

...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ...The recently established case Matter of Dhanasar provides the three-prong test that US Immigration officers.... As Immigration is a federal law, Casseus Law provides legal services to people located in all 50 states...

...overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification (Option 2)...Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/978-1-62703-764-8_9. PubMed. More Resources at Addgene:  ...customized to direct assembly of DNA fragments. When designed correctly, the recognition sites do not appear... scheme is as follows: the gene of interest is designed with Type IIS sites (such as BsaI or BbsI), that...and do not appear in the final construct. The destination vector contains sites with complementary overhangs...ligation can be done in one 30-minute reaction. The destination vector and entry vector(s) are placed in a single...IIS enzyme and ligase. Although the original destination vector + insert may spontaneously religate, this...

...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...biology – they can deliver materials to specific destinations, haul a variety of cargo, and even handle a ...delivery of the actual virus to which immunity is desired. Instead, components of the virus are now delivered...