...colleagues. Here you simply PCR-amplify your vector and your insert, DpnI digest away the templates, and transform...will require a PCR reaction or two to generate templates for the cloning process. Time spent actually doing...relatively new to the cloning game, however, or simply looking for a more streamlined approach, you might...Golden Gate assembly allows one to simultaneously complete an ordered set of scarless ligations following...1. Li, Chaokun, et al. "FastCloning: a highly simplified, purification-free, sequence-and ligation-independent...

... et al., 2015). In these studies, the strategy employed was to deliver a standard AdV vector to the targeted...AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells yielded...genes. For this you would need to provide a DNA template to repair the double strand break via homology...Cas9/sgRNA and one vector for your specific DNA template. Using this two vectors system, Manuel Gonçalves...a potential platform that could be realized via simple switching of the relevant gRNAs and donor DNA Adenoviral...tools can also be used to deliver Cas9 and DNA templates in somatic cells of adult animals in order to ...vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array...

...thought of as descendants of totipotent cells. An example of pluripotent cells is embryonic stem cells (ESCs...lineage. Blood (hematopoietic) stem cells are a good example of multipotent cells. Unipotency: As the name suggests... mostly from abnormal and/or supernumerary preimplantation embryos that are produced during in vitro fertilization...maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells found in the bone marrow...in science communication and publishing after completing her postdoctoral training from Massachusetts ...Extraction of Pluripotent Embryonic Stem Cells: Implications for Regenerative Medicine and Reproductive Medicine...embryonic stem cell lines from IVF embryos after preimplantation genetic diagnosis. Hum Reprod, 2008. 23(1):...

...some simple solutions available for researchers to deal with all of these problems. The simplest and best...forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment...cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might think EGFP is unlikely... a cell. Unfortunately, things are not quite so simple. Concentration is the number of molecules within...around that fusion protein is very high. Real world examples of these situations in cells are described below...form heterodimers (i.e. EGFP and mCherry). These simple strategies can help you make better fluorescent...

...and replaced it with a double-bonded oxygen. This simple enzymatic reaction enabled conversion between the...and one-ring pyrimidine base. This is a far more complex chemical task, not a job for a single enzyme. ...bind DNA, Cas9 requires (1) a guide RNA, (2) a complementary DNA sequence, and (3) a protospacer-adjacent...separate and preventing repair based on the complementary strand. This could explain why the edited U ...original G is not primarily converted back to a complementary C. Optimized base editing with eOPTI-CGBE and...so the Yidi Sun and Erwei Zuo labs tested and implemented several of these advances for CGBEs (Yuan et ...replaces a target region with a newly synthesized, templated sequence. And the story still isn’t over! Keep...

...his program. Initially, Provost made his labs completely open-ended, allowing every student to work on... the culmination of many such experiences. For example, students in Provost’s course recently used a combination...Kowalski et al., 2016; Scott, 2021). In fact, completion of Provost’s CURE increased students’ confidence...Community (MCC) that seeks to reduce barriers to implementation. Funded by the NSF (with Co-PIs Jessica Bell...for your classroom, I highly recommend you try implementing one. It's likely only a matter of time before...Poirier, V., Pellettieri, J. 2020. The exon junction complex is required for stem and progenitor cell maintenance... Kowalski, J., Hoops, G., Johnson, J. 2016. Implementation of a collaborative series of classroom-based...

...many biological fields. In synthetic biology for example, high-throughput cloning can be used to combine...one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent... variety of other clever applications. In one example, modular constructs generated with MXS-chaining...being directional, meaning that (in the previous example) one can control whether the third module is added...various cloning methods are listed in Table 2. For example,  chaining-based cloning methods require that the...general types of modules can be assembled. For example, because CpG dinucleotides are underrepresented...these types of constructs robustly, quickly, and simply. We hope that you can similarly apply MXS chaining...

...the other screen, implying that ORF and CRISPRa libraries can be used in complement to study gene function... stranded DNA breaks that commonly result in a complete loss of function while nuclease-deactivated Cas9... in the depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello even with only...have several limitations. CRISPRko results in a complete loss of function meaning that if an essential ...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature517.7536 (2015): 583. PubMed PMID: 25494202...et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...collaborations, and commitments to and proof of compliance with the grantor’s requirements as well as state...expertise on the processes needed to successfully implement, manage and close-out a grant. Grant professionals...reports are submitted and project evaluations are completed. To enter and advance the profession, you can ....S. Bureau of Labor and Statistics shows that employment of grant writers is expected to grow by 16% between...grant by another state. With the blessing of my employer, I took on my first consulting job, and I began...mentoring. How do I become a grant professional? Employment opportunities for those with expertise in grant...application writing and development, project implementation, program evaluation and grant management.  ...

...enChIP system enables us to express the CRISPR complex much easier in various cells, including primary...technologies consisting of iChIP and enChIP are recent examples. We also developed the inducible translocation...interference-PCR (ORNi-PCR) method to suppress PCR amplification of specific DNA sequences. Thus, we've made ...system of journals and grant applications, for example. However, at the same time, it's fun to be involved...professional activities because I can contribute by, for example, helping to improve the papers submitted to journals...give to post-docs and graduate students as they complete their appointments in your lab?  Fujii: These ...develop new technologies, hopefully they'll be completely different from what we are now doing in my lab...

... used as the first letter of a plasmid name and simply denotes that the object is a ‘plasmid’. “p” is ... empty backbone name in your plasmid name. This simple piece of information can often convey many important...insert abbreviation to specify what species it is. Example: ‘h’ is for Human (homo sapiens), ‘m’ is for mouse...plasmid and their relative position to the insert. Example, if you have a Flag tag on the N-terminal of your...directly before or directly after the insert name.   Example: If the Glutamine at position 295 was mutated to...pBACKBONE-Flag-hGene-EGFP or pBACKBONE-Flag-hGene(wt)-EGFP These simple rules allow any scientist to know what is in a ...

...appropriate time—for example, the cell must have enough nutrients available to complete the entire round ...and regulation in bacteria Before looking at an example plasmid, let's review how E. coli normally replicate...chromosomal replication. Adapted from BioRender template. Since DnaA binding is necessary to initiate... stringently regulate plasmid replication. For example, the plasmid ColIb-P9 encodes the protein RepZ ...Inc, also encoded by the ColIb-P9 plasmid, is complementary to part of the sequence forming this pseudoknot...replication. At high copy numbers, RepA proteins form complexes between iterons on different plasmids in a process...

...whole-genome/transcriptome” studies, in which, for example, the activity of all genes in response to an experimental...the globe (Nüsslein-Volhard et al., 2012). One example (which I will come back to) is the one-eyed pinhead...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...morpholinos. When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either...genome is useful for a variety of applications. For example, specific tissues such as blood cells or neurons...regulatory systems have also been successfully implemented in zebrafish. Developmental biology insights ...well-suited for neuroscience investigations. For example, neuronal activity can be tracked in whole brains...

...fields in order to provide useful solutions to complex challenges. As a cross-disciplinary researcher,...Many of the challenges in science today are too complex for any single discipline to solve them on their... in terms of communicating ideas. In 2012, for example, 3 billion hours of video were watched each month... disciplines leading the way, the future of complementing published research with video overviews was ...when it is of quality). On the site you can, for example, learn about how researchers are using “big data...contribution. It offers a 5-step process and several examples for creating effective research videos.  Karen...derived from research. The platform uses video to complement traditional print journals and expand our ability...

...certain loci to create changes in phenotypes. For example, CRISPR activation has been used to differentiate...gRNAs targeting every gene in the human genome for example, can be made easily and cheaply using oligo library...change the sequence targeted by the activator by simply providing a new gRNA rather than engineering an...allows for exploratory-based research as well. For example, CRISPRa can be used to find potential cancer drug...transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038/nature14136...Kadoch C (2018) Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale... (2014) A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging....

...coming weeks, but, in this post, we will outline a simple method for selecting the guide RNA, validating ...CRISPR Design tool supplied by the Zhang lab. This simple website invites you to input the target region ...happy with your selection, ordering the sgRNA is simple and easy. Several companies supply gRNA sequences...you gRNA in vitro first. In vitro screening is a simple and fast method for validating your selected gRNA...base pair sequence surrounding your gRNA. Once amplified, you can insert this region into a pCAG-EGXXFP...). Testing the efficacy of your gRNAs is then a simple matter of transfecting HEK293T cells with your ...perfect primer sets to use to genotype your pups. A simple PCR with these primers will let you know if there...

...undergraduate experiences, they were provided with ample resources and a positive perspective on the transition...career path of their choice. This failing becomes amplified by systemic biases which, in the absence of support...from all areas of the scientific training and employment infrastructure. A Stakeholder Workshop at Janelia...career training approaches in a way that can be implemented widely in the community.  Talk alone is not going...resulting in concrete actions and outcomes. For example, pd|hub assembled a coalition of higher education...Sciences (NIGMS) will support the development and implementation of collections of evidence-based practices....

...confrontational situations with others because you simply cannot see things from their point of view and ...herself to the nearby village (and to the reader) by simply listening. Chances are, you know someone like this...teams resolved similar issues in the past, or how completely different teams are tackling comparable conflicts...it will be important to retain the stories and examples that have guided your resolution processes in ...collaboration may not be the easiest resolution to accomplish, but there is a potential for long-term gains...into your conflict management skills. It’s also completely OK to recognize that conflict resolution may ...or are looking for a promotion, have some solid examples of times when you have used your empathy, listening...

...this tradition. We now have over 30 full-time employees, so we created one long lunch table and we eat...about your colleagues on a personal level, and employees often cite lunch as one of their favorite parts...provide free food on special occasions. However, employees regularly bring in baked goods and we have a few...To-Do list long enough that there is no hope of accomplishing it all. How do we encourage people to take a... Hack Days, and we’ve seen great success at accomplishing “we-should-really-make-time-for-that” type projects...projects. Cross-team “Random Lunches” With more employees, Addgene has naturally divided into smaller functional...

...sensors Combinations of classical genetic tools, for example Cre/loxP or Flp/FRT, with AAV vectors are common...vectors, and post-translational regulatory elements. Examples of these approaches are given in Tables 3-7. 3...specificity of cell targeting even further. For example, injecting AAVs at one location can result in expression...that received information from a region. In the example shown in Figure 1, injected retrograde AAVs at ...neuroscience is becoming more and more versatile. To complement the wealth of genetically encoded tools available...only reagents, but also method information. To complement these efforts, we’re beginning to share data ...