...rounds of replication are initiated before the cell has divided. This means that genes located near the...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...genes closer to the origin of replication will be overrepresented compared to those further away. As a...maximal gene expression near genomic origins of replication, the stabilized promoters created by Segall-Shapiro...the control of IPTG. Because RepA is a plasmid replication initiator protein, they could alter copy number...

...digital notebook and make sure you note the freezer location of every plasmid in your collection. Use one of...access and use the materials they need, and avoid duplicating your effort. In addition, if you should choose... important step in ensuring quality, as it will catch any errors made during the cloning, sample handling... for more tips on sequence analysis, plasmid verification, and using NCBI BLAST. Tips for high-throughput...result against the expected sequence for quick verification, which can be done through the align tab of ... plasmid verification...

...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ...nickase variant, lowering Cas9 expression, and truncating gRNA sequences used for targeting; however, these...on-target and off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time ..., 98% of TdTomato expressing cells were GFP+, indicating a robust, but specific system. T-DDOGs also successfully...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Could Cas9-DOG be developed soon to allow genome modification only in a small subset of cells? Other proteins... PMCID: PMC4096992. Find plasmids from this publication at Addgene. 2. Tang, Jonathan C.Y., et al. “...PubMed PMID: 26258682. Find plasmids from this publication at Addgene. 3. Kirchhofer, A., et al. “Modulation...

...choose well. What are their qualifications? Do they have certifications? How can they demonstrate their...  Check out Joanne's Reddit AMA I am a strong advocate for peer mentoring, but volunteer mentors are ...help you. A good coach usually will be a clear communicator and make direct suggestions for improvement ...coaching is nice if you can get it.  Because I am located in Cambridge, MA (and because we have a lot of ...she is especially excellent in coaching for communication skills. Industry transition is also an area ...

...experiences to undergraduate students (1, 2), but educators face significant challenges in designing suitable... become very different due to a whole genome duplication and extensive rearrangements in the S. cerevisiae...overexpression vector. This plasmid contains origins of replication and selectable markers that allow students to...met deficiencies (manuscript in preparation), indicating that Met protein functions have been largely ... research into undergraduate courses and in communicating science to the public. You can find out more...). Vision and Change in Undergraduate Biology Education: A Call to Action, Washington, DC. 2. President's...

... biological reagents have a ton of data on the catalog page. It’s not always super easy to navigate the...originally described in a peer-reviewed scientific publication. We love science, but we don’t have the resources...questions about an Abcam antibody. Have your catalog number ready. I've attempted to answer several ...Addgene support, make sure to mention the plasmid catalog number, or at least the plasmid name in your initial...be fairly fast, getting the full answer to a complicated question may take some time. If the customer ...message Answering customer service questions is a delicate balance of knowledge, patience, and diplomacy,...

..., its dimeric nature can interfere with some applications. Several labs have been working to produce monomeric... for molecular fusion and membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038...library generation and screening that led to identification and characterization of AAV.CAP-Mac. Image ...rodents and broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467...proven itself a good option for essentially all applications. Addgene’s plasmid collection includes over ...clone (M2) has been widely used in a variety of applications. Find anti-DYKDDDDK recombinant antibodies here...

...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...expectation and comparison on the antibody signal’s location. If a distinct imaged pattern does NOT align with...literature and is unrelated to the target protein, we categorize the antibody as having non-specific binding in...

...scientific poster apart? First, a good poster should catch the audience’s eye and draw people in. That can ... sections should be as concise as possible, communicating only the most important results to your audience...have the advantage of being able to verbally communicate your work, so you don’t need every little detail...may choose to display it as rows. It can be more catchy because it is generally less common and will stand...advantage of being different and potentially more catchy, but it can be trickier to make figures fit nicely...relevant to the work you are presenting. The latter is catchy, but beware. A distracting background or clashing...

...chemical replication of RNA in the absence of evolved enzymatic machinery (i.e. RNA replication with no...Guangzhou and the Science City The conference was located in the modern, relatively new “Science City” area...and technology companies and start-ups choose to locate there. I made time to go into the city of Guangzhou...inheritance that might be less error prone than DNA replication. He too mentioned how “surprising” small RNAs...Steitz quipped that RNA in the ribosome is in a complicated conformation “not like DNA which has a boring...

...targeting anywhere in an exon. But things get more complicated if you’d like to use homology directed repair...They created a selection phage (SP) to carry catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase...test xCas9 3.0-3.13, Hu et al. first restored catalytic residues D10 and H840. To ensure that the clones...loci displayed a broad range of PAM sequences, indicating they may be less predictable than SpCas9 off-... that we have only scratched the surface of modifications that can be made to Cas9. It is a pleasant surprise...editing rates. This PAM flexibility should expand applications that require very specific targeting, like homology...

...antiviral defense). Cascade is brought to the target location using a gRNA and can then recruit Cas3, which ... type I, the most abundant type in the Class 1 category. These type Is can then be further broken into...of DNA much like a fisherman would reel in a big catch. It was originally suspected that Cas3 moved a couple...Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome...unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-...Koonin, E. V. (2015). An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology...

...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do...more cells, while AAV can be titrated following purification using qPCR or digital droplet PCR. Insect cells...to collect your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in...titer the virus, or go straight into your viral application, like protein production. Titration of baculovirus...Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation (AAV); centrifugation (lenti...

...RESCUE in therapeutic applications, the authors tested various dRanCas13b truncations that would allow packaging...longer stretches of duplexed RNA. Using an ADAR2DD catalytic mutant, they showed that editing is mediated by...into adeno-associated viral vectors, they tested truncated ADARDDs to see if they could shrink the construct... to deaminate cytidine and used dRanCas13b (catalytically inactive Cas13) to target the cytosine deaminase...construct for viral delivery. They found C-terminal truncations allowed the same or improved editing ability ...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...of a non-living viral particle, and how we as dedicated researchers can exploit it. This blog was written...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral...acid of the Bryan strain of Rous sarcoma virus: purification of the virus and isolation of the nucleic acid...

... Aptamer biosensors are used in a variety of applications, including detection of environmental pollutants... opens aptamers for use in many entirely new applications including aptamer based fluorescent biosensors... render this approach less sensitive in most applications. Learn About Fluorescent Biosensors Light-up..., MG proved to be too toxic for many in vivo applications, but this work proved the concept that RNA could...and biochips. Ultimately, there are just some applications that lend themselves to aptamer-based instead...Kyung-Mi, et al. “Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed...

...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

... they are based on light which is very easily scattered by tissue. Ultrasound, on the other hand, is a...protein nanostructures called gas vesicles, which scatter sound waves and thereby produce ultrasound contrast...limiting their applicability. They also did not scatter ultrasound nonlinearly, making them difficult to...out to develop next-generation bARGs that could scatter ultrasound nonlinearly and be easily expressed ...promoter pBAD and a low-copy number origin of replication (p15A) worked best under a wide range of inducer...smaller genetic footprint, and nonlinear acoustic scattering. The best candidate for this was the Anabaena...

...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...