...thermophiles, bacteria must be grown at lower temps. This approach only works for facultative thermophiles (high ... occurs? This is different than the engineered approach used to make eSpCas9 and SpCas9-HF1. By mining...SpCas9 RNP’s and a guide targeting a site with an overlapping PAM. In both cases, GeoCas9 and SpyCas had similar...could also be used with in vitro molecular bio applications that require high temperature reaction conditions...always, the plasmids are available from Addgene. Happy CRISPRing! References Harrington, Lucas, et al....

...scientific aspects of writing. Following his advice, I applied to the class. Receiving the email that I had been...the ASM headquarters. I decided to go ahead and apply; thinking I probably would not get selected for ...knowledge with practical understanding of how to apply it. The personal interaction of critiquing others...well as being critiqued added an extra level of application not present in the online class.   Four ways ... manuscript should be able to be enjoyed and appreciated by all fields not solely the specific area the...

...learn how easy this process is and to get tips for applying it in your own lab. Table 1: Comparison Between... E. coli and transformants selected with the appropriate antibiotic. Although the precise mechanistic ...additional 15-17 bases that are complementary to the overlapping vector ends. See an overview of the PCR process...an agarose gel. You should see bands with the appropriate sizes for your vector and insert. Once you’ve...correct product. Once you find a colony with the appropriately sized product, purify and sequence the plasmid...

...tag to remain on your protein for downstream applications Below, we describe a few of the most common...not to perturb protein function for downstream applications. Tag cleavage must be performed after primary...also provides a visual detection mechanism via applications such as western blot and immunofluorescence....dimerizes in solution, which can affect downstream applications. Also, purifying GST by affinity chromatography... are generally best suited for their primary application. Ready to start tagging? We are here to help ...

...isolating the cell line all the way to modern applications. What the HEK is it?   HEK cells were first...cell biology, there are thankfully many other applications that they are useful for! What the HEK is it...cell line and we will highlight some of those applications here.   Virus production HEKs are the go-to ...proteins at high levels. In a case where clinical applications of recombinant proteins are necessary, HEKs ...significantly reduces the cost of production. Research applications There are two areas of research where HEK cells...

...NgAgo replace CRISPR? Such a drastic shift won’t happen overnight, but there are a few reasons why you ...flexibility in choosing a genomic target. NgAgo also appears to outperform Cas9 in GC-rich regions. DNA guides...staggered (Cpf1) cleavage. NgAgo takes a different approach, randomly removing 1-20 nucleotides from the cleavage...site specified by the gDNA. Depending on your application, this could be favorable or unfavorable. In a...activity. One-guide faithful NgAgo guide loading appears limited to the period when the protein is being...

...generation of various flux imbalances. The conventional approach has typically been to either systematically delete...networks and multiplex gene control. However, these approaches are often met with the challenges of regulatory... possible. From a design perspective, this is appealing since the RNA scaffold modules are comparatively... multiplex control via scRNA design is readily apparent in metabolic engineering. The authors elegantly...control of gene expression. This strategy can be applied to effectively change metabolic flux. The dCas9...

...axial dimension that will appear in focus). This means that the images will appear sharp without interference...excite a molecule of GFP. Because red light has approximately half the energy of blue light, two photons of...density of red-photons be establish for this to happen.Using red light has a second advantage: red light ...objects lying within the diffraction limit will appear as one object in the final image. Over the past...of light – think of a single GFP molecule – will appear as a fuzzy 200-300 nm spot when imaged. Super-resolution...

...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down...disrupts protein function, an N-terminus tagging approach should be pursued, however, clonal lines must ...me at [email protected]. Keep pipetting and happy genome editing! Pooran Dewari is a postdoc in Steve...optimizing CRISPR/Cas9 genome-editing methods and applying them to study brain tumour biology. When not pipetting...

... of applications with the potential to save lives. While most scientists recognize and appreciate the ... Yamanaka himself, during his keynote address, appeared almost incredulous that the field has exploded...daunting because they have no clear cut answers, I applaud the stem cell community for addressing these important...but they are also the reason this research is happening. Stem cells scientists have certainly not lost...

...have high levels of off-target effects, both of happen with Cas13. CRISPR-Csm degrades both nuclear and...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...fluorescent proteins (FPs) necessary for many of these applications have lagged. Fixation, chemical staining, and...well-suited for the demands of advanced microscopy applications such as super resolution, electron, and expansion...

...interactions. Beyond these common applications, researchers are finding new ways to apply fluorescent proteins everyday...Zhou and Fatima Enam. There were many excellent applicants but Xin and Fatima stood out for their creativity...Light-inducible Proteins (FLiPs) Fluorescent proteins can be applied in a long list of creative ways. In her graduate...utility of these FLiPs in a variety of research applications and particularly notes their ability to be precisely...

...altering its function). HDR is crucial for certain applications, for example, correcting the allele that causes...this question led us to develop a new, rational approach to improve the efficiency of sequence replacement...introducing a cut, with residence times on DNA of approximately 6 hours. To our surprise, we measured identical...region allowed sequence replacement rates of approximately 1%. This is by no means a large number, but ... order of magnitude greater than transfection approaches. Moreover, the cost of reagents is relatively...

...together a grant application? Are you familiar with the publishing or patent application processes? How ...How can you stand out among all the candidates applying for the position?   Here are a few things to consider...makes sense for your skills, and the job you are applying for. Also remember that a resume is typically ...have done is closely related to the job you are applying for, in which case talk about your research, as...

...next-gen sequencing. ChIP is an incredibly useful application for exploring these interactions, and can give...including epigenetic modifications. But it's not an approach for the faint of heart!  The Process ChIP can...preferred method, enzymatic digestion can be appropriate when your protein of interest is a low abundance... upfront investment than many other antibody applications, this investment can well be worth it once you... idea to add "patience and an extra-methodical approach" to the top of your protocols when embarking on...

...advances and how some of these variants may be appropriate for your specific research question. Considerations...off-target activity, which may be undesirable in many applications. At the time of our first blog post on choosing...Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created pX-evoCas9, which...enzymes may better suit specific experimental approaches. One example is CasX, characterized by the Oakes...While no enzyme is perfect for all experimental approaches, knowledge of both the advantages and limitations...

... by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants in bacteria...the key components of EvolvR and its potential applications. Challenges of existing mutagenesis methods...exist, but there are a few drawbacks with these approaches that makes it challenging for users to achieve...is capable of mutagenesis in E. Coli, but this approach requires special bacterial growth conditions in...more than one mutation near the target site. Applications of EvolvR The Dueber Lab used EvolvR to discover...

...multicolored nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion...furimazine. In addition to the previously described applications, the enhanced signal of eNL allows their use...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially where photobleaching...concern, these constructs come in very handy. This approach is especially suited to complement optogenetic...Therefore, especially thick samples may not be appropriate for use with Nano-lantern technology. Multicolor...

...disciplines. In biology, innovative new imaging approaches are constantly being developed which combine ...biological protocols and revolutionary physical approaches. However, with each new technical achievement... greatly increases accessibility. Through this approach, the Planktoscope, a portable device for studying...on-going creativity with the construction plans. Appropriate open-source licenses for hardware (e.g. CERN ...optical setups. References Joel T. Collins, Joe Knapper, Julian Stirling, et. al, "Robotic microscopy for...

...principles we used in our daily content writing applied to alt text. We also were aware of the need for...they hoping to get from it? The image in Figure 1 appears on our protocol page for AAV Purification by Iodixanol...a lot of jargon and have a technical, detailed approach. Other times, it is more helpful to use plain ...start small with alt text or immediately dive into applying these four steps elsewhere, engaging in this practice...from posters to papers.  We hope this helps, and happy writing!   Resources Additional resources on the...