...or A much further from the ‘NGG’ PAM cannot be editable? They can, but they require other base editor ...about CRISPR, one question probably comes to mind: can CRISPR be applied to fix the defective base precisely...CRISPR-mediated base editing (BE), these types of repairs can now be done more efficiently than before. A base ...guanine (G)? You may wonder if a CRISPR-base editor can perform this change. However, there are no naturally...successful evolution of an adenosine deaminase that can act on DNA for adenine base editing. They first created...pKABE6.3, pKABE7.9, and pKABE7.10) so that the user can optimize base editing efficiency based on the location...to the protospacer adjacent motif (PAM). ABE7.10 can edit target 'A' if it lies in a small genomic window...

...cases, counter-selection escape can occur: single crossover colonies can acquire the ability to grow on...multiple transformation and plasmid curing steps, and can generate off-target mutations. Modifying bacterial...alternative, endogenous homologous recombination enzymes can also accomplish the desired genomic modification....crossover”) which results in a merodiploid. This can be accomplished by selecting for an antibiotic resistance...on sucrose will identify double crossovers. This can be an efficient method to generate precise, unmarked... because in these circumstances, the conjugation can be so inefficient, that instead of the resulting ...the scarless modification of bacterial genomes. It can accomplish large deletions, insertions, and even ...

...master mix of enzymes can be purchased from a company (e.g. NEB or SGI-DNA), or can be mixed yourself (e.g...easy assembly of multiple linear DNA fragments, but can also be used in basic cloning of an insert into your...great part about this mix of enzymes is that they can all work at the same temperature, so the entire reaction...Miller Lab Protocol). The Gibson assembly process can be used to assemble up to 6 fragments in one step... required homology between neighboring fragments can be created via PCR amplification with primers that...fragments. However, by using Gibson assembly, you can insert both the gene of interest and the tag sequences... Excluding the time it takes to get primers, you can have your construct made in 5 days. One drawback...

...blue. Students can create synthetic riboprobes off of linearized DNA templates which they can easily generate...differ. For example, Girardia sp. and Dugesia dorotocephalacan can be fed dsRNA-expressing E. coli that are...version of L440. This vector is versatile in that it can be used for both in situ hybridization and dsRNA-...bidirectional transcription to create dsRNA. Students can produce DNA templates via PCR off of the same plasmid...comparison. Performing WISH using these plasmids can result in  the labeling specific cell types or entire...number of genes that other classrooms and researchers can easily investigate in planarians. References 1. ...Kimberly C. Tu, Alejandro Sánchez Alvarado, The American Biology Teacher, Vol. 79 No. 3, March 2017; (pp...

...researcher can cause them to interact by stimulation with blue light.  Similarly, light can also be used...further probe cellular function. The CRY2clust Plasmid can be found here. Park et al. Nat Commun. 2017. PubMed...“addicted” E. coli strain expressing this operon can serve as a living biosensor for measuring caffeine...pDCAF3 operon, the growth of this knockout strain can be rescued by the xanthine produced via complete ...organization. The design use to detect PKA activity can be generalized to measure phosphatase activity, second...proteins have improved photophysical properties and can be used in quenching, FRET (Förster resonance energy...

...drop-down and contact Addgene. We can only accept plasmids that can be used under BSL1 conditions.  ...deposit spreadsheet?  Our plasmid deposit process can be broken down into four simple steps: Send plasmid...most involvement with Step 1, and, while data entry can be a chore, we’ve made it easy to send us your plasmid...others need to be filled with short-answer text. You can also directly paste sequence data into the appropriate...rabbit), synthetic, or other. If other, the species can be indicated in the “Species Other” column. Relevant...QUEEN-generated GenBank files (Mori and Yachie, 2021) but can accept sequence files in any format. Distribution...replication, choose the Pir1 strain. If your plasmid cannot be propagated in any of these standard strains,...

...of accumulated wisdom so that you, the customer, can get the most out of your email or phone call. Though...saying “Hey look! This stuff works!” These papers can be a particularly good resource if you are considering...Hi! We ordered something from you and it’s wrong. Can you help us fix it? Please address as soon as possible...begin my responses asking for more specifics. We can help you faster if we get as much information as ...least the plasmid name in your initial query. This can reduce the number of messages in our thread or remove...but a picture of a gel or a sequencing trace file can go a long way in helping us understand the nature... be courteous. We understand how exasperating it can be to lose time and money on a potentially faulty...

...split-Cre systems which can take up to 24 hours to cut, Opto-Cre-Vvd is fast and can cut in as little as ...utilizing recombinases to manipulate genomes, scientists can control expression of their genes of interest. While...expression tools in and of themselves, their activity can be fine-tuned and precisely controlled through the...sensitivity to ambient light, and versatile as it can be activated by both low and high-intensity blue ...single active site. The RuvC active site in Casɸ can both process crRNA and carry out the crRNA-guided...human and plant cells. Casɸ from Biggiephage can eliminate competing mobile genetic elements. Image...

...cloning (discussed below) can run on the order of $20-30 per reaction but can be used to quickly generate...fragments in a prescribed order. These fragments can be pooled in one tube and ligated in as little as...needed for this method is relatively inexpensive and can be obtained for about $70, resulting in a price of...dollars per reaction. By comparison, Gibson assembly can cost over $15.00 per reaction if you purchase a pre-made...remember that commercially available cloning kits can often make the process much more streamlined, because...homology based methods, like Gibson or SLIC, since one can pool the components and define their final order ...

... light exposure can also lead to cellular phototoxicity, and experimental success can be affected by both...biologists over the last two decades, but their use can be limited by a few caveats. Since fluorescent proteins...and showed that these three luminescent proteins can be used together in cells. As Nano-lanterns are not...fluorescence, so higher concentrations of the compound can be added to the media. Cellular esterase then converts...optogenetic tools, where light used for excitation can cause unintended activation of the optogenetic system...proteins (FP) require external light activation, you can’t use fluorescence to monitor processes directly affected... physiology in your system. Optical sectioning cannot be conducted with luminescence imaging since there...

...protein interacts with that one promoter can tell you, it can be easy to get lost in the details. The ...like stem cell biology where questions of ethics can have profound impacts on the development of applications...and appreciate the importance of ethics, the topic can often take the back burner at scientific conferences...international borders. While questions of ethics can be daunting because they have no clear cut answers...stem cell community with their research, I think I can speak for Addgene when I say that I'm glad we're ... important questions that the scientific method cannot address. Listen to Our Podcast Interview with Stem...

...differentiation To understand how each human TF can contribute to differentiation, the Zhang lab transduced...Similar to the Human TFome Library, the MORF library can be used with other selection and screening mechanisms...library webpage. Disease models Good disease models can often be difficult to generate, in part because of... dysregulation. A targeted TF screening approach can not only lead to a better understanding of disease...simulate a disease in a model system. TF screens can be designed to identify custom TF programs to acutely...and GFP fluorescent controls. Alternatively, you can order individual plasmids from the library to further...Individual cellular fates were then matched with candidate TFs responsible for that differentiation.   ...

...Addgenie expertise we can. We even have a Slack channel just for asking people if they can write or review ... is our Help Center, help.addgene.org, where you can easily search articles answering all of our frequently... our depositors and customers are asking. So how can you get involved? Keep asking your burning questions...and tell us what you want to know more about! You can also suggest blog topics or even reach out about ...support and advance the scientific community, and we can only do that with your help.     Additional Resources...

... been motivated to do anything and everything we can to help find a cure -- and quickly. Normally drug... approach is to discover key human proteins that can be targeted (hopefully by existing therapeutics that...these experiments, there's so much to do that we can use the whole science community's help. You could... but one are in a pLVX vector background, so you can package them into lentiviral vectors if you need ...FedEx guy has been the best: Thanks, Todd! As you can imagine, it’s also been quite the administrative [email protected] with your order number and so it can be prioritized. ...Arabia -- as well as closer to home in the US and Canada. In fact, we received so many requests (175 and...

...genome. Thus the gRNAs responsible for a phenotype can be identified by next-generation sequencing of the...readout. This retroviral mouse CRISPR pooled library can be used for screening of cells that are refractory...lipoproteins through the circulatory systems, but can also lead to the formation of atherosclerotic plaques...transmit electrical information. Such voltage imaging can be performed in intact animals and offers many advantages...found several voltage biosensors and conditions that can be used to monitor real time voltage changes in worms...-mOrange and MacQ-mCitrine. All of these sensors can detect action potentials in worms with fluorescence...libraries was not possible. Primary murine T cells cannot be transduced by lentivirus and have been difficult...

...protocol can save weeks of trial-and-error. A recommendation for the correct statistical test can save a...Spain and works in the field of genetics and cancer. You can follow her on Twitter at @mahesa2504       ...community where both brief and long-term collaborations can be easily started and easily credited. And nothing...scientific research. It’s completely free, of course. You can find more information about us and what we do in ... based in Spain and works on climate change. You can follow him on Twitter at @IgnacioAmigoH     María...while most of the world was locked down, a South African company wanted to test if a type of antibodies ...

...attorneys with a biology background were in demand. Q: Can you tell me a bit about your job currently? What ... the business world from the mindset of a lawyer can bring a new perspective to an organization. I'm used...an organization. I think these are things that I can bring to Addgene -- that comfort with saying, “if... and judgement to the organization and see how I can help. Q: What do you enjoy doing outside of work?...

...the CTD can get cozy with DNA. S. pyogenes Cas9 recognizes the PAM sequence 5’-NGG-3’, where N can be any...for genome engineering, and studying its structure can help us understand the parameters of CRISPR experimental...Other techniques, like electron microscopy and FRET, can offer more information on these regions.   Cas9’s...reflecting the less stringent recognition mechanism that can tolerate mismatches in this region. The crystal structure...any base. In Figure 5, we can see why: two arginines make base-specific hydrogen bonds to the guanines...paired to each other? At this point, the protein can switch between the closed and open conformations ...with the rest of Cas9 and is highly flexible; it can rotate about 140 degrees between inactive and active...

...expertise and skills of many of my colleagues, I can also orchestrate the formation of high-functioning-but-temporary...to have their own specialties. This type of team can exist indefinitely. If you’re looking for this type...biostatistician, and that one golden-handed technician who can do a certain difficult assay with her eyes closed...the skills are not cherry-picked. We take what we can get since participation on such a team is strictly...without such an association, start one! These teams can offer a sense of solidarity you’re going to crave...about this.” Sometimes the common roles of a leader can be successfully doled out to multiple people: someone...people actually like meetings, but run well, meetings can be a critical part of any successful team. For reasons...

...depicted on the left) is ~5.8 kb in length. The plasmid can exist on as a dimer, depicted on the right, with ...bacterial cells compared to monomers, although this can be prevented via antibiotic selection (Crozat, 2014...instances, please note that the supercoiled dimer band can sometimes overlap with the band corresponding to ...Electrophoresis (CGE) analysis Another technique that can detect multimers is capillary gel electrophoresis...laser-induced fluorescence (CGE-LIF) for detection and can provide data on the quantity of various topologies...strain that is deficient in recombinase protein RecA can reduce the frequency of multimerization, and in some...temperature such as 30°C or optimizing the incubation time can help maintain cultures in log phase growth. In a ...