...encouraging user contributions (using simple web forms) while also maintaining a relatively strict database design...This post was contributed by guest blogger Talley Lambert, a Research Associate at Harvard Medical School...design of an imaging experiment.  The choice is non-trivial, however, as FPs are tremendously complicated entities...on a web-based interactive visualization of FP attributes (which turned into www.fpvis.org).  That page...and we very much appreciate even those small contributions). Of course, no community-editable database ...simplifies the process of building advanced multi-parametric search queries.  Looking for a bright, monomeric...frequently) for the sake of comparison or ease of retrieval.  These collection pages can be kept private, ...

...their destination co- or post-translationally. TRICK: Visualizing the first round of translation While... co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging by Coat protein Knock-off...RNA-binding proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change...RNAs appear red. To enable this color switching, TRICK requires the following components: a GFP protein...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important...important in development, and Halstead et al. used TRICK to examine translation of Drosophila patterning ...patterning gene oskar. Early in development, the osk-TRICK reporter was double labeled, indicating translational...

...fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside...which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have...They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on....that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment.... If you inserted these two pieces of DNA by restriction enzyme cloning, you would have to do it in two...the smaller overlapping sequences left with a restriction digest. A study in 2013 found that Gibson assembly...PCR step difficult--OR there is no convenient restriction site for linearization. This is a perfect case...

...permission to distribute. Distribute pending QC: your plasmids become available for distribution as soon as...archive any plasmids you've deposited with us and distribute them to scientists worldwide. What's more, depositing... pre-publication plasmids" and set a desired distribution status. You can submit "Published plasmids":...description of the deposit. Select your desired distribution status: Hold for publication: we will initiate...additional questions. Bacterial Strain Addgene can distribute your bacterial strains if they: are E. coli ... +1 (617)-ADDGENE (233-4363) if you’d like to distribute your bacterial strain(s) through Addgene. Description...information for the gene or insert. Step 4: Growth and Distribution Please indicate which strain Addgene should ...

...that plasmid deposits come from 49 countries* and reached 102 countries. We started from 474 plasmid deposits...deposits coming from two new countries bringing the total to 51 countries with plasmid deposits!  ...Twitter!  That’s today through Friday. There will be trivia, prizes, and more! We wanted to take a week to ...impact in open science. Depositors consistently contribute to our collection so that we can offer the scientific...been requested about 1,500 times from labs in 45 countries*. While the bulk of these plasmid deposits came...you are a depositor or not. We will have Addgene trivia (with prizes!), polls, and shout outs, and we’ll...the schedule for the week: Monday: Fun stats and trivia begins! Tuesday: Depositor features (plasmid deposits...

...Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  Listen to the anti-CRISPR podcast...fluorescent voltage indicator Archon Article contributed by Shreya Vedantam  Listen to the voltage indicator...segment The neurons in the brain compute using electrical impulses to coordinate emotions, thoughts, and... behavior. Historically, scientists study the electrical activity in the brain by inserting electrodes...thousands of proteins for one that can report electrical activity via imaging. They took the light-sensitive...easier with a 2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning...pUC57-L3L2) that offer the advantage of using restriction digest cloning to introduce the GOIs into the...

...anode/iblot gel matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper...remain intact and the protein is separated through a matrix by charge. In a denaturing western, the protein...smaller molecules moving more quickly through the matrix. For the purpose of this blog, we will be focusing...confirm the size of the protein of interest. An electric current applied to the gel causes the negatively...proteins encounter less resistance in the acrylamide matrix and migrate faster through the gel. For better ... In addition, PVDF membranes can be repeatedly stripped of an antibody and reprobed with different antibodies...sandwiched” together with blotting paper and an electric current is applied causing proteins to migrate...

...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....

...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...

...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...

...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...