... CRISPR Plants CRISPR Plasmids: Plants Browse CRISPR Plasmids By Function...have been designed for use in plants. CRISPR...following CRISPR plasmids have been designed for use in plants. Cut Fully functional CRISPR/Cas enzymes will introduce... Marker PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted single-strand breaks in...strategies reduce unwanted off-target effects. Nickase mutants can also be used with a repair template to introduce...

...Rinehart Lab Phosphoprotein Reagents Rinehart Lab Phosphoprotein Reagents Protein phosphorylation is one... for Improved 2015 Phosphoprotein Reagents (DOCX, 525.9 KB) Reagents (ca. 2014) described in: Enhanced...Protocol for Reagents (ca. 2014) from the Rinehart lab: Users Guide for 2014 Phosphoprotein Reagents (PDF, 691.3...the Rinehart and Issacs labs at Yale have made improvements to this system by genomically recoding E.coli...translation system (pSerOTS-C1* (V70) 188537 ). These reagents are described in detail in (Mohler et al., Mol...for laboratories performing phosphoproteomic experiments in need of reference spectra or phosphopeptide...phosphopeptide standards for quantitative assessments. The mode #1 phosphosite library can be generated with either...

...describing these reagents are provided below: Zinc Finger Genome Engineering Reagents Zinc Finger Arrays...OPEN) Reagents - Engineer zinc finger arrays via OPEN method Modular Assembly Engineering Reagents - Engineer...Find reagents for engineering and expressing zinc finger proteins deposited by Zinc Finger Consortium... Zinc Finger Consortium Zinc Finger Consortium Reagents You may also like... Keith Joung Lab plasmids ...Daniel Voytas have deposited at Addgene various reagents for engineering and expressing zinc finger proteins...Genome Engineering Technologies TALEN Engineering Reagents CRISPR Plasmids...

...function in zebrafish, plants, and human cells... Kits Zinc Finger Consortium OPEN Reagents Zinc Finger Consortium...Consortium: OPEN Reagents (Kit # 1000000013 ) Depositing Labs: Keith Joung This kit consists of plasmids...plasmids Detailed Information Protocols & Resources Contents of Kit Original Publication Oligomerized pool ... request from the Joung lab) and the following reagents (listed below and available individually or as...as an “OPEN Accessory Reagent Set”). These reagents are also valuable research tools for using the bacterial...used to engineer ZFNs that function in zebrafish, plants, and human cells ( Maeder et al., Mol Cell 2008...

...consist of two components: a short ~100 nt guide RNA ( gRNA ) that uses 20 variable nts at its 5’ end ...additional pages describing Joung Lab CRISPR-Cas/RGN reagents: Cpf1 expression plasmids (AsCpf1 and LbCpf1) ...) for use in mammalian cells SpCas9 variants with no detectable off-target effects Modified PAM specifity...

...Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z, Joung JK. 2016. High-fidelity CRISPR-Cas9...Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JR,...specialized Cas proteins snip foreign DNA into small fragments approximately 20 bp in length and paste them into...These edits can include mutations seen in human patients, protein tags, or loxP/FRT sites, among others... High fidelity Cas9s: Three high fidelity Cas9 variants , SpCas9-HF, eSpCas9, and HypaCas9 display very...Streptococcus , Streptomyces , and others) Drosophila Plants (monocots and dicots) C. elegans Yeast ( S. cerevisiae...

... Protocols Weighing Reagents Weighing Reagents Protocol Intro to the Lab...how to obtain the correct amounts of materials for an experiment or reagents for a stock mixture is an...basics of how to weigh reagents. Protocols...get started in the lab! Introduction For many experiments, you’ll need to make buffers, media, or other...of this task is making sure you’re weighing all reagents precisely to create the solution you expect. Understanding...essential laboratory technique, as imprecise measurements can affect the outcome of your experiment. In...protocol, you will learn the basics of how to weigh reagents. Wear the appropriate personal protective equipment...

...cleavage sites 12 nt and 13 nt away from the target site rather than the optimal <10 nt distance. Employing...Oligonucleotides) Use 100 nt homology arms for large insertions (long ssDNA; IDT Megamer® ssDNA Fragments) Test both ...configurations with varied nick distances (40 - 130 nt). From these results, it’s clear that editing is ...efficiency is very low when the gRNAs are too close (7-23 nt nick distance). Both D10A and H840A are potent editors...they observed >20% repair (max 27%) across the 51 nt region using either a top or bottom strand ssODN ...insertion (mCherry) using IDT Megamer long ssDNA with 100 nt homology arms. Schubert and Yan next examined a ...target site, they designed a D10A nickase strategy (46 nt nick distance, PAM-out) to see if they could solve...

...Transparency Another major benefit of preprints is their openness. Preprints can be read freely by anyone and...review or curation models where editors’ comments, reviewers’ comments, and author responses can be shared ...of the benefits of preprints. Some worry about faulty work being posted as preprints, assuming that peer...the manuscript prior to submission. Preprints at Addgene Preprints are all about sharing scientific knowledge... position working with preprints in life sciences. He also posted two preprints of his own research during...ASAPbio, Preprints and Rapid Communication of COVID-19 research: https://asapbio.org/preprints-and-covid... the results of the work. Yes, those control experiments are important - but without letting other scientists...

...of CRISPR components, new Cas9 variants are being discovered all the time and the requirements of these...or Cas9 mutants developed in a variety of labs (see table below) have different PAM requirements. These...your experiments. Use the following flow chart to get you thinking about the types of experiments you'...describe the functions of the various CRISPR/Cas9 components. Please note that while this post is intended... thereof. This individual protein has all the components necessary to: 1. Bind to a guide RNA The guide...These different PAM requirements allow researchers to target many different genomic loci. 3. Cleave target...resulting in a double-strand break (DSB) Cas9 and its variants have two endonuclease domains: the n-terminal ...

...are reached, how do science labs celebrate accomplishments and their team's hard work? Do succesful scientists...work-life balance. How do you celebrate your lab’s achievements? Tom Ellis highlights the importance of acknowledging...acknowledging his teams successes. “When PhD students graduate there has to be champagne, that's a given...and see if we should start celebrating papers and grants too with pizza or pub.” The Cepko Lab celebrates... the lab. The Church Lab celebrates successful moments in the lab with parties in the lab’s common areas... aren’t locked into a schedule.” George Church comments on how he would go home in the afternoon during...interesting things from children, their classmates, and parents”. He also cooks and cleans with his family, “sometimes...

...devoted to the other affinity reagents.  Affinity Reagents  Affinity reagents are molecular tools that have...affinity reagents.    Types of Affinity Reagents  (Listed in no particular order.) Fab fragments are created... affinity reagents deserved to be celebrated, there are many other amazing affinity reagents we just didn't...alternative affinity reagents" - everything from camelid antibodies to antibody fragments to tools completely...reagent?  The main advantage of alternate affinity reagents is their size. Antibodies are large enough that... a barrier at times. Smaller, slimmer affinity reagents can have deeper penetration into a tissue, useful...antibody. In imaging applications, smaller affinity reagents means higher resolution. Many of the below are...

...than some of the larger fragments.  Think of antibody fragments as new components in your antibody tool ...example, Fab fragments are molecules composed of just the Fab portion of the antibody. Fab fragments are useful...available different fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced...110 kDa. Fab' - These fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge...independent Fab fragments with small tails of the hinge region. Size: ~55 kDa.  Fab - These fragments are produced...results in two independent Fab fragments. Size: ~50 kDa. Fv - These fragments are composed of just the variable...antigen binding fragments. Created with biorender.com.   Why Fabs?  Antibody fragments have a handful...

...and standard molecular neuroscience validation experiments we discussed last week are only half of the battle... but the primary one is that some behavioral experiments require stimulation only under certain contexts...More recently, wirelessly-controlled optogenetic implants have been reported by several groups [4, 5, 6]...much greater movement flexibility in behavioral experiments. However, the technical demands of using these...interest, it’s time to get to the fun stuff: the experiments! In our final post in this primer on optogenetics...considerations when performing optogenetics behavioral experiments. Thank you to our guest blogger Derek Simon! ...

...contributed by guest blogger Derek Simon. The actual experiments you do will be determined by the topic you’re...about when developing optogenetics behavioral experiments. There are far too many behaviors that have utilized...sufficient readout to proceed with your behavioral experiments. Comparison of c-fos amongst animals that receive...[6]. Technical differences include the ferrule implants and patch cables. One huge advantage of using ...their superiority as behavioral models. Beyond rodents, optogenetics has even been applied to other species...Materials Science of Optogenetics Experiments ... optogenetics behavioral experiments...

...pointing out a group of students in a university hall but not knowing if these students know or interact with...considered when analysing data from your FRET experiments. You may have to design several different constructs...to target mTurquoise2 to various subcellular compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses mammalian...and Venus Other issues that can affect FRET measurements include: the brightness of a fluorophore pair...required to make this method useful for dynamic experiments in which FRET changes are large. Two other methods...is destructive and cannot be used for dynamic measurements. Extra care should be taken so as not to destroy...imaging method, limiting its use in many FRET experiments. In addition, other environmental factors, such...

...order, my top 10 open science developments, 2013 edition. 10 new developments in open science 1)      In ... share your favorite OA developments of the past year with us in the comments. Or let us know your favorite...minutes to reflect on some of the most noteworthy developments in open science this year. Here it goes, in ...stewardship of clinical trial data, serve society and patients, and respect industry concerns.” 5)      In the...aftermath of the Supreme Court’s decision to ban gene patents in a case against Myriad, an organization called...

...replication components are called helper plasmids. Below, we’ll breakdown the vector components first by ...there are bonus components to these vectors that aren’t reflected in the common components outlined above...lentiviral guide outlines these components.     Fig. 1 - Viral vector components of retrovirus  Adeno-associated...production reasons, multiple plasmids with many components are used to package lab grown viruses. It can... protocols! In reality, there are many shared components across viruses (AAV, lentivirus, etc.) and their... what gag and pol stand for, we demystify all components of viral vectors here! Need Virus? Check out...adenovirus, and adeno-associated virus (AAV). The components of these vectors are also split across several...

...errors in the design of cloning strategies set experiments back days or even weeks. Primer design was done...principles. For every task, we envision what the user wants to do and make the path to accomplishing their goals...is an ongoing process that involves iterative refinements in response to customer feedback. An example ... and display frequently used genes and control elements. Development of this tool required creating a ...variant of a feature. Instead, we identified common variants, and then crafted a detection algorithm that tolerates...sequence has changed. Identifying plasmid control elements Coding sequence features are relatively straightforward...straightforward to define, but for control elements such as promoters and transcription terminators, the boundaries...

...through two sequential homologous recombination events: The plasmid carrying the desired genetic modification... Partial Characterization of the Pink and Blue Pigments of Pocilloporid and Acroporid Corals. The Biological...G, Kühl M, Hoegh-Guldberg O (2000) Fluorescent pigments in corals are photoprotective. Nature 408:850–...