...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...

...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...

...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...

...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...

...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–...

...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...

... the field of grants. What is a grant professional?  In the past 5-10 years, the Grants Profession has...collaborations, and commitments to and proof of compliance with the grantor’s requirements as well as state... for grants, the amount of grant funds available and the increasing regulatory process for grants has ...grant consultants who offer grant training, mentoring and coaching services, including Smartegrants, to ...choice exam.  The bottom line on being a grants professional The Grants Profession is in a rapid growth phase... Inc., a national grant consulting firm, and Smartegrants, a grant professional development firm The path...established grant professionals is learning to succeed in grants through trial and error, through winning and losing...

.... Now is the time to make bold statements. While some of the statements may be slightly far reaching, ...consider the reviewer's comments. Whether the proposal is funded or not, the comments help gauge room for ...government and academic institutions. New graduate students may assume that their advisor alone applies for... allot some if not all grant duties to trusted students. For example, if the proposal call aligns with...write a grant!”, “I don’t have time for this if he wants data by next week!”, and “This is his job!” filled...various skill building opportunities that graduate students and postdocs should actively pursue. Proposal ...italic, or underline font formats helps these statements stand out from the rest of the text. Specific...

...single 96-well plate for HDR that satisfy all requirements. To alleviate this bottleneck, we began building...whole or in part. For example, you can analyze experiments designed outside CrispyCrunch, or you can input...step of mutating guide sequences in a way that prevents re-cutting but does not disrupt gene expression...that worked with NGS data were: Crispresso CrispRVariants AmpliCan We chose to adapt Crispresso by Luca...re-create a match for the active guide. With these constraints in mind, we implemented the following algorithm...Design donor RNA Design primers Review and order reagents Perform your wet lab experiment Analyze the data...high-throughput design and analysis of CRISPR experiments. It has certainly boosted our productivity in...

...(i.e. the plasmid complements the deletion). If the phenotype is corrected, students can be relatively... multiple enzymatic steps (5). In their experiments, students test whether or not proteins from the fission...extensive rearrangements in the S. cerevisiae lineage (7). In our class research project, students transform...in other experiments (Figure 2).   Complementation reveals conservation of function Students have used...Boston College. Students in the course work in teams of three in sections of 15 students. Within each section...also used to teach students about restriction mapping and western blotting. Students who participate in...authentic research experiences to undergraduate students (1, 2), but educators face significant challenges...

...levels and affect behavior. A fact to consider: Rodents can hear ultrasonic frequencies (> 20 kHz, produced...

...around the globe, we’re now sharing over 100,000 reagents including plasmids, ready-to-use viral preps, ...

.... Both awardees emphasized the contribution of students in their labs and the importance of the strong... what can be done with this technology to help patients. Dr. Stoffels and others in the audience discussed...technologies. Addgene is proud to be helping get CRISPR reagents out to academic labs. It is our hope that robust...