...often doubles the volume of culture used for inoculation if we get inadequate plasmid DNA yields. Why ...growth conditions, and the material used for inoculation. To further complicate matters, plasmid copy ...may lead to loss of the plasmid, and using older plates could increase plasmid loss or mutation. Let it...these factors are generally, but not perfectly, correlated. Typical growth times for high copy plasmids...smaller cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration...

...in mind! This section of our plasmid pages is relatively new, so some of our older plasmids may not have...publication. Addgene makes this easy by providing a template citation for you to use in your Materials and ... Use the Addgene catalog number to easily find template citations for Addgene plasmids you have used in...on the N or C terminus of the transcribed and translated insert. If it is an empty vector with a tag, ...here. Articles Citing this Plasmid: This is a relatively new feature of our plasmid pages, and provides...

...not narrow enough to distinguish between closely related colors. Furthermore, most FPs  have a broad range...oxygen deprived environment. Recently, a new GFP isolated from the Unagi eel was shown to mature independently...chromophore. This can be from a few minutes after it is translated to a few hours. For example, superfolder GFP ... of how bright is the emission. Brightness is calculated as the product of extinction coefficient and ...College of Medicine. He is interested in everything related to gene expression, particularly at the RNA level...

...rather “let’s see how we can make this fun activity relate to STEM.” Figure 1 shows the activities we had ...initial guidelines I received from the teachers, I formulated the presentation as follows (which could be adapted...molecular mechanism, I was careful to keep it relatively simple. I honed the complexity of the talk by...moving along much faster than it can be officially regulated. I did not try to sugarcoat the potential dangers... on a kid’s face when you’ve shown them how to isolate DNA from a strawberry, or demonstrated that they...

... thing if CRISPR hadn’t made manipulating the genome cheap and relatively easy? Have all of these things...unlikely to be surprised by many of Zimmer’s revelations. The issues he discusses have been making the...before then. Other areas are newer, such as the relatively recent resurgence of “Do-It-Yourself Science”...its own Franklin in Doug Prasher, who originally isolated the GFP gene but was not included amongst the ...

...visualize PPIs that regulate numerous cellular functions, including ubiquitinoylation, kinase signaling,...generate negative control constructs — more on this later. For more on constructing fusion proteins, check...necessary because split FPs can associate, albeit at relatively low frequencies, without being brought into close...visualized makes it amenable to studying PPIs under relatively physiological conditions when compared to FRET...

...fluorescent proteins, promoters, enhancers, polyadenylation signals, inducible gene-expression sequences...generate the four initial unique constructs and later combine all four constructs into one 15 kb insert...polycistronic containing multiple identical polyadenylation signals or promoters Gibson et al., 2009 ...”) can be readily reused in the assembly of any later construct (i.e.,functional cassettes are recyclable...Neveu PA. MXS-Chaining: A Highly Efficient Cloning Platform for Imaging and Flow Cytometry Approaches in Mammalian...

...future edits. In contrast, since Cpf1 cleaves relatively far away from the PAM, NHEJ might retain the ...confirmed. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Comparing Cpf1...throughout the 23-base sequence. Double mismatches ablated Cpf1 activity, except when they were present in...exciting both in terms of basic science and translational applications. This discovery of this type V ...we had a lot more to learn about CRISPR biology. Later work, like the adaptation of Cas13 to RNA targeting...

...usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon... Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used ...crystallizing it or testing its function. Small ubiquitin-related modifier (SUMO) can help with folding and stabilization...and purification An affinity tag, generally a relatively small sequence of amino acids, is basically a...Combo Consists of a bacterially derived in-vivo biotinylation signaling peptide (Bio), flanked by hexahistidine...

...do a lot of DNA work but never get to actually manipulate the organism they study (unless if you count ...study of diverse organisms. Methods for genetic manipulation still need to be developed for each organism... currently for terrestrial frogs. Cnidarian-Dinoflagellate symbionts (Virginia Weis) Sea anemone Aiptasia...Henry) This black footed slipper snail can be manipulated by injected RNA and can express fluorescent proteins...is developing tools for pan-arthropod genetic manipulation. The ReMOT control system (Receptor-Mediated...

...peripheral blood mononuclear cells (PBMCs). PBMCs isolated from whole blood present many benefits for the...patients are reprogrammed less efficiently, no such correlation has been found between the age of the patients... amplification in the host cell. This enables relatively high and long-term expression of the reprogramming...using PBMCs as the source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of...Blood Cells: An Efficient Protocol Using Serial Plating of Reprogrammed Cells by Centrifugation. Stem Cells...

... to grow on plates or in liquid culture to amplify the library, followed by DNA isolation. The amplified...screening-scale transduction of cells, resulting in cell population mutated by the viral pooled library. These cells...payload is adequately represented in the cell population, you’ll need to infect many more cells than the...Joung J, Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F. Genome-scale...

...PE6b was very efficient for short, unstructured templates and significantly reduced undesired edits (indels...complex edits, such as long or highly structured templates. Cas9 variants PE6e–g often had even higher editing...potential to reduce negative side effects. This platform offers a promising approach for experimental therapies...illuminated with blue light, catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential... et al. (2023). Engineered allostery in light-regulated LOV-Turbo enables precise spatiotemporal control...

...in your favorite neuronal population and track connections to that population. Limitations of common genetically...to define those that target specific neuronal populations in the brain. The Allen Institute and the Janelia...also been important in mapping, monitoring, and manipulating neuronal circuits. Epitope tags are short antigenic...cells. They can be used for protein localization, isolation, and tracking experiments (Rizzo, 2009). With ...

...Secretary of Labor Thomas Perez updated the overtime regulations of the FLSA. The exemption salary was set at ... postdocs are at each of these institutions (populations potentially from 1 to 5761, according to the ...research on these institutions, we can say with relative certainty that just over 40% of current postdocs...for academia Changes are afoot for the postdoc population, and it is unclear how this will all shake out...effects the new laws will have on the postdoc population. In the current funding climate combined with...

...Having successfully navigated the trials and tribulations of the process, I’ve provided guidance and mentoring...interviews but no job offers, is a failure to clearly articulate one's talents and ideas. Some key points from...question? This should be something that anyone could relate to, that people would agree is important to understand...provide the first defined model system that we can manipulate to study pathology and therapies." This is...interview, your previous success will not necessarily translate into getting a job. Your papers and pedigree get...

...strategy for increasing the size of the AAV donor template for CRISPR-mediated homology directed repairs ...acceptor of the transgene to help prevent the accumulation of such truncated proteins. Examples of use...homologous recombination approach delivers large donor templates using two sequential homology directed repairs... the gRNA target site that mediated the donor template’s integration so that the site is reconstituted...which results in the integration of a larger donor template.  Drawbacks: Transgene expression varies with...

...can do and, in this way, science has always been related to government and, consequently, politics. More...Inclusion of data informing policy specifically related to the following subjects: Climate change Vaccines...Healthcare Science education To make scientists relatable (17, 20). This is an effort to show the general...all you need to do is select your state. State Legislatures: To find the website to contact representatives...Lawmakers need to use a scientific approach to formulating views Pew Research Center: Public and Scientists...

...factor fusions modulate iPSC reprogramming efficiency. Listen to the segment on modulating iPSC reprogramming...Hot Plasmid 3: Regulating telomere length with TZAP. Listen to the segment on regulating telomere length...

..., generating new therapeutics, and sometimes formulating new biological concepts. However, rates of reproducibility... However, even simple procedures like pouring a plate or running a gel have intricacies that aren’t always...have plans to expand to other protein technologies later. Organizations such as BenchSci and FPbase are working...find information about research tools. BenchSci collates information about antibodies while FPbase curates... soon, the Center for Open Science does host a platform for pre-registration wherein researchers can submit...