...fundamental steps: creation of the RNAi trigger delivering the RNAi trigger characterizing the RNAi phenotype...This post was contributed by guest blogger, Robert Orr, who recently received a Ph.D. in Biology and ...complementary to your gene or genes of interest to trigger RNAi activation. A typical RNAi experiment can ...RNAi-based investigation. Creation of the RNAi trigger requires a few initial considerations, such as ...artificial microRNA). Using a DNA template for the RNAi trigger is the most prevalent method due to ease of use...assortment of siRNAs generated from a long hpRNA trigger, only a single ~21-nt interfering RNA is produced...precursor mentioned above and is a popular RNAi trigger in the mammalian research community. Fortunately...

...Comparison Between Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning...This post was contributed by guest blogger Meghan Monroy, a graduate student in Protein Science at the...elements of biomedical research. Traditional restriction cloning is one of the oldest of these techniques...purification, ligation, and transformation. While restriction cloning is still routinely performed by many ...Cloning Choose Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - ...products and digest with methylation sensitive restriction enzyme DpnI to remove the templates. Digestion... in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis....

...researchers in 45 different countries — and as of 2024, we’ve distributed over 100,000 viral vector preps...adeno-associated viruses (AAVs), will know just how tricky these tools can be to use. AAVs, which can target...repository each year from 2016–2023. B) AAV preps distributed per year by Addgene from 2016–2023. Tools...requested (Figure 3). Over 400 biosensors were distributed in 2023 alone. You’ll also find a large number...from 2016–2023 by type of tool. B) AAV preps distributed by Addgene per year from 2016–2023 by type of...AAV preps for the seven most popular serotypes distributed per year by Addgene from 2016–2023. How to... A) GCaMP6, jGCaMP7, and jGCaMP8 viral preps distributed per year compared to other AAVs preps from 2016...

...three tabs.  Each of the three tabs - Contributions, Indel Distribution and Traces - provides particular details...analysis screen. ICE analysis details Contributions   The Contributions tab shows the inferred sequences present...the most 5’ cut site.     Indel Distribution In the Indel Distribution tab, you’ll find an Indel plot ...This article was contributed by Jessica Roginsky, Scientific Support Lead at Synthego. Article source... useful measure to determine how many of the contributing indels are likely to result in a functional ... interested in understanding how many of the contributing indels are likely to result in a functional ...plot (left panel) which displays the inferred distribution of indel sizes in the entire edited population...

...attL-containing entry vector.   Method C: Restriction cloning of a restriction enzyme fragment containing the DNA...scientists are no longer limited to traditional restriction enzyme cloning. Instead, you can choose a molecular...Figure 5: Method C to create an entry clone: Restriction cloning the inset into an attL-containing entry...integrity of your expression clone via sequencing or restriction digest! Then, you can transform or transfect ... day, as opposed to 2+ days with traditional restriction and ligation cloning. It is also possible to ...vectors. The cloning process is simple -  no restriction, ligation or gel purification steps are required... generate entry clones by TOPO cloning or by Restriction Cloning Destination vector attR sites for recombination...

...James Trimmer of the University of California Davis School of Medicine on this grant. Dr. Trimmer has created... ready-to-use-proteins to facilitate use. Dr. Trimmer’s lab will ensure the quality of the antibodies ...Addgene to bring this resource to life,” says Dr. Trimmer. But in order to create such a resource, we need...on being financially self-sustaining through distributions of research materials, such as plasmids and ...new project to reach a sufficient number of distributions to cover costs, which can limit expansion into...Initiative has been funding laboratories, such as Dr. Trimmer’s lab, to create recombinant antibodies for use ...https://doi.org/10.1038/520295b Bradbury ARM, Trinklein ND, Thie H, Wilkinson IC, Tandon AK, Anderson ...

...Deposit and Distribution Agreement establishes the terms under which your material will be distributed. In signing... of distributing under Addgene’s Industry MTA (IMTA), which allows the plasmids to be distributed for ... 784,795 MTAs with 8,798 organizations in 111 countries. From 2019 to 2023, we averaged facilitating 89,477...actually own the majority of the materials we distribute; therefore we are not a party (signatory) to ...re not! The vast majority of our plasmids are distributed under the UBMTA, which is widely used across ...organization agrees to let Addgene receive, store, and distribute your material under the specified terms. This...depositing institute that decides the terms of distribution for their plasmids. MTAs at Addgene Since Addgene...

...This post was contributed by guest blogger Nathaniel Roquet, a PhD student in the Harvard Biophysics ...Once expression of a particular recombinase is triggered, its effects on the state cannot be undone and...predominant state of registers within a sample using a metric that we describe in the Materials and Methods section...only semi-quantitative. It won’t tell you the distribution of states in a sample; instead, it will suggest... of registers in a sample based on a similarity metric that we defined in our paper. In our opinion, the...attB-attP pairs on a register to record all permuted substrings of a set of chemicals. Specific examples of arrangements...arrangements that should be able to record all permuted substrings of up to seven chemical inputs are given in Table...

...relies on Type IIS restriction enzymes, first discovered in 1996. Type IIS restriction enzymes are unique...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method has...unique from "traditional" restriction enzymes in that they cleave outside of their recognition sequence, ...to as MoClo, this strategy uses the Type IIS restriction enzymes BsaI and BpiI/BbsI to efficiently assemble... Gate protocols, complete with helpful tips and tricks, see The Sainsbury Lab website or Engler & Marillonet...destination vector contain multiple internal restriction sites that may not be amenable to "domestication...cloned DNA as tandem multimers using class-IIS restriction enzymes. Lee JH, Skowron PM, Rutkowska SM, Hong...

...unique restriction enzyme site and unique primer sites for more rapid screening by restriction digest ...for genetic optimization of industrial yeast strains. However, industrial yeast strains are more difficult...CRISPR library Addgene has had the privilege of distributing several human and murine CRISPR pooled libraries...single-tube, 3 day process by utilizing pre-assembled trimers to reduce the number of cloning steps. The FusX...PMID: 26787909 EasyClone 2.0 Yeast Toolkit Many industries, such as the food and beverage industry, the ...transform. To increase the genetic toolbox for industrial yeast strains, the Borodina Lab has recently ...et al. Nat. Methods 2009. PubMed PMID: 19915560 TRIBE: Targets of RNA-binding proteins identified by editing...

... on versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to ... But...how? Cloning is no longer limited to restriction enzymes - although many labs still love using...your E. coli do the work for you. Traditional restriction enzyme and ligase based cloning is a relatively...The ligase cost is comparable to that of the restriction enzymes and overall you’re looking at a price...of a few dollars per reaction for traditional restriction cloning. Sequence and Ligation Independent Cloning...appropriate inserts by PCR, there is no need for restriction digests, gel purifications, or ligations. The...efficient due to the use of the versatile type II restriction enzymes. In Golden Gate cloning, the desired ...

...Gelsinger in an adenovirus-based gene therapy clinical trial gone horribly wrong. It brought the whole field ...presence of antigens (such as viral vectors) without triggering a destructive inflammatory immune response. At...would take another 6 years before the 1st clinical trial started, and 10 more years to obtain FDA approval...natural serotypes are currently used in clinical trials, a variety of engineered ones have shown great ...in preclinical studies and could reach clinical trials soon. The AAV safety profile is excellent and ... cells using a lentiviral vector and a clinical trial is ongoing. Moving forward with ocular gene therapy...AAV-based ocular gene therapies is impressive: clinical trials for choroideremia, X-linked retinitis pigmentosa...

...This post was contributed by guest blogger Katrin Michel. Cre-lox is an incredibly popular and powerful...times. Many thanks to our guest blogger, Katrin Michel! Katrin Michel is a Postdoctoral Researcher at MIT...pair palindromic repeats separated by an 8 bp asymmetric core. Recombination between these sites can lead...discovered that it is not the exact sequence of the asymmetric target site core but its 8 bp length that is ...plasmids and the low concentration of recombinase restricts their expression to individual cells. With this...knockout in the same mouse.   Scientists always strive for more. In order to analyze the function of individual...Through a partnership with genOway, we are able to distribute materials containing FLEx technology. Read the...

...level, read on for more in-depth tips on probe chemistries, primer optimization, controls, and analysis....Picking your chemistry  The two most common qPCR chemistries are hydrolysis probes (which you probably know...commercial name: Sybr). However, there are other chemistries available and they can be the more optimal choices...choices at times. For example, chemistries that rely on hairpin design of probes/primers often offer higher...stable than the hairpin bond.  Alternatively, chemistries that combine the primer and probe into a single...comparison of features of several commonly used chemistries)   Chemistry Background noise Probe ...gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design...

...studying the brain in a way that goes beyond such restrictions, you may want to consider a systemic capsid ...enhanced function after intravenous administration, distributing widely throughout the organism to target large...and recombination target sequences, denoted by triangles] facilitate tissue or cell type specificity. An...ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal ...your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb) for your...promoters and enhancers (Mich et al., 2021) can help restrict expression to your cell type of interest, while... with Cre-expressing transgenic mouse lines to restrict transgene expression to specific tissues and cell...

... a SmaI or XmaI restriction enzyme digest (the ITRs contain SmaI and XmaI restriction sites). Another ...these tools, I found myself writing AAV production tricks into the margins of my protocol. While these tips...recombination of duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary...ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of different concentrations...during AAV production is overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected...day. Refer to the AAV Iodixanol Gradient Ultracentrifugation Protocol for more details. Pro tips Watch...titer AAV. Do you have any AAV production tips or tricks? Please share them in the comments below! Special...

...This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such...limitations through the use of engineered Type IIS restriction enzymes and careful choice of junction sequences...involves the simultaneous activities of a Type IIS restriction enzyme such as BsaI, and a DNA ligase such as...receives the assembly after similar Type IIS restriction enzyme cutting results in 5’ four base overhangs... provide optimal reaction temps for both the restriction enzyme and ligase, thereby enhancing the ordered...destination construct and functioning Type IIS restriction enzyme and DNA ligase, any single omission should...

... This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means... assumed that the molecules are homogenously distributed throughout the volume and can freely tumble in...be much higher than if they are homogenously distributed and mobile throughout the cell. If two copies...CFP, YFP, BFP, Cerulean, Turquoise, Venus, and Citrine) can be monomerized with an A206K mutation [2].... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing the lowest...

...research by banking and distributing monoclonal antibodies at cost. We distribute over 65,000 antibody units...The following post was contributed by the Developmental Studies Hybridoma Bank. Antibodies are the most..., is a repository for monoclonal antibodies, distributing over 3,000 antibodies and associated hybridoma...the University of Iowa. The minimal fees from distribution funds our operation. Antibodies are glycoproteins...PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies against popular fluorescent...Posts about Scientific Sharing: The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years...

... media platforms are available or used in all countries; not all areas of any given country have equal...can guarantee you that either resource or time restrictions (or both!) will be a major limiting factor at...flexibility to visualize and contextualize. Can be distributed in digital or analog media formats. Cognitively...platforms can splinter audience. Primarily digital distribution (though radio is possible.) Written works...works Low Flexible. Low barrier to entry. Ample distribution channels both digitally and physically. Easy...overwhelming your audience. Digital or print distribution.  Static. Can be difficult to convey an entire...highlighting, or omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific...