...DNA, the CTD is also sometimes called the PAM-interacting (PI) domain. The guide RNA is bound primarily...most likely through unstructured electrostatic interactions. It also can’t recognize PAMs, so it associates... less stringent recognition mechanism that can tolerate mismatches in this region. The crystal structure.... As mentioned above, however, Cas9 sometimes tolerates mismatches in the next 8-10 bases after perfect...this region of Cas9 also allows the protein to tolerate RNA:DNA mismatches between base pairs 18 and 20...Each new structural insight also spurs the next generation of engineered CRISPR enzymes with their own tradeoffs...Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization...

...the conference After using your elevator pitch several times at the conference, you might notice some ...larger side? At large conferences, chances are that several talks will be going on at once in different rooms...a conference, be sure to practice beforehand. Generally, two types of people will visit your poster: those...have “tweep ups” where you can meet those you’ve interacted with on science Twitter in real life. If there...the tweep up (and for the entire conference, in general). After the conference Review your favorite conference... considered networking, but here are some more general tips. Before the conference Networking is key even...

...that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in human disease...and is stable and active for greater than 50 generations. Figure 1: Mobile-CRISPRi's modular components...highly conserved glutamine-fructose-6-phosphate aminotransferase (glmS) gene in the recipient bacteria due ...Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease (S. aureus... library strains was measured after 6 and 12 generations with or without induction of CRISPRi knockdown...

... copy number variation (CNV) assays. There are several varieties of these, but in this blog post, we're...length of the amplicon. As the probes can have several different markers, it can also be used to run multiplex...Do you need to call exact copy numbers or just general trends? How confident do you need to be in your...Addgene blog   Droplet Digital PCR for AAV QuantificationPolymerase Chain Reaction Overview and Applications...Applications Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer...SYBR Green I and TaqMan quantitative real-time polymerase chain reaction methods for the determination ...

...experiments require some consideration of FP properties. It is important to look out for general problems associated... judgement by simply “looking at them”. Modern cameras and image analysis software are highly sensitive...microscopy cannot be interpreted as direct molecular interaction without further investigation. One fluorescent...microscopy method commonly employed for analyzing interactions between two molecules more closely is Förster...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

... (cap) genes. AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded...consuming to prepare the 40% PEG solution. PEG takes several hours on a heated stir plate to dissolve and requires...carefully layered and then the viral suspension generated during AAV production is overlaid. Following ultracentrifugation... comparing to an AAV standard curve, which is generated from an ITR-containing plasmid. This whole process... always be included. While the physical titer generated by qPCR is useful, I test a few different concentrations...inverted terminal repeat sequences. Human gene therapy methods, 23 1, 18-28. PubMed PMID: 22428977. 2....

...incubation at 55 °C - a dangerous, non-physiological temperature for mammalian cells and one which diminishes ...could detect knock-in of tags at a target site. Overall, 64% of researchers were unhappy with the new method...the water. On the bright side, successful indel generation and epitope knockin was achieved by a few respondents...ssDNA leading to inefficient downstream editing. Overall, the real take-home message thus far, based on ...form a complex with a 5’-P-ssDNA guide at room temperature. Until that time, those who have been lucky with...Pooran Dewari is a postdoc at the MRC Centre for Regenerative Medicine (CRM), Edinburgh. His research interests...

... Their focus at the lab is on plant-pathogen interactions, and her aim is to produce constructs and edit...biosensors designed to elucidate the molecular interactions that go on between plants and their pathogens... share and educate and enable plant science in general. We are funded by a charity and since we are making...elements anymore. The kit contains sequences for several different fluorescent proteins. Visualizing fluorescence...piece of DNA. We do a lot of protein-protein interaction work and we’ve gotten some tools back from Addgene...Twitter account recently inspired by an academic in literature who gave a talk to the women’s research network...

...infection; however this would not be great for a gene therapy vector! For viral vectors, only the protein for...viral vector particles are made while missing the polymerase gene may seem like a mystery. But it’s simple...transcription and translation. Here, we will discuss several aspects of the viral “life cycle” in order to understand...step 3), but note also that it is worthwhile to generalize the idea of “transduction” to mean any transfer...et al., 1973). Interestingly, retroviruses are generally only “pseudodiploid” in that — while they have... M., & Bishop, J. M. (1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis...

...incorporated a discovery-based lab on planarian regeneration into one of my own courses, I was excited to...phosphorylation on the enzyme’s structure, function, and interaction with other proteins.   It is important to...be authentic – students should be attempting to generate new knowledge that will be of genuine interest...and bench work to identify regions of MDH that interact with citrate synthase and presented their findings...increasing diversity and inclusion in STEM disciplines (Bangera & Brownell, 2014; Duboue et al., 2022), as they...classroom.   References and Resources References Bangera, G., Brownell, S. 2014. Course-based undergraduate...