... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool... efficiently, as those that do not contain the desired insert should not grow.   Figure 2: Image...

... as the OG (as the kids say) affinity reagents deserved to be celebrated, there are many other amazing...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...°C and can refold correctly after denaturing.  Designed ankyrin repeat proteins, or DARPins, are genetically...a wide variety of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells. They...

...molecular biology experiments was in the job description, but this skill is not just about reacting to...puzzling through your broader research questions and designing future experiments. Being able to work through...research non-academic careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined...most likely cause, you implement an experiment designed to fix the problem. Then you assess whether your...

...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...   Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral Tet-on ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...Strong protein-protein interactions with other peptides. Created with BioRender.com.     Many biologists... conjugation. But hopefully, this introduction provides a good base from which to dive deeper!References...

...another. If you are using barcodes (and you should be!) bear in mind that your barcodes are being applied to...containers, not to the contents. Tightly coupling barcodes and containers might not be such a bad idea, tightly...Resources on the Addgene Blog Learn about How We Use Barcodes in the Lab Get Tips on How to Keep a Lab Notebook...

... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...Addgene partnership to provide AAV viral preps, includes listing of transfer status for viral preps....vectors See Addgene's Current AAV inventory , which includes all viral vectors transferred from the Penn Vector...

...newer generation lentiviral packaging systems were designed to prevent the formation of replication competent...use 3 plasmids; an envelope plasmid that usually encodes VSV-G, a packaging plasmid containing structural...transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for larger ...the best titer possible. While this information provides a great starting point, it may not translate to...

...soCoChR-expressing system. Taken together, Shemesh et. al. describe a novel system for the precise mapping of neuronal...the single-cell level. Various soCoChR plasmids described in this work including the soCoChR-GFP plasmid...information about spatial and temporal organization. The design use to detect PKA activity can be generalized to...choice for FRET assays where a dark acceptor is desired due to its very low quantum efficiency. ShadowY...

...tools, they do have undesired off-target editing effects. ABEs have been described to introduce base changes...efficient. Informed by these mutations and previously described TadA mutations (Gaudelli et al., 2017), they modified...heterodimeric fusion of wild-type TadA and TadA7.10, they designed one set of ABE8’s similarly (ABE8.x-d). In order...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...

... developed based on 100 structure-guided spike designs and has improved protein yields and stability. ...Plasmids deposited from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding...Liu Y, Chou C-W, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang N, Amengor D, ...Finkelstein IJ, McLellan JS (2020) Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science...

... They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an “assist peptide” that...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but ...recombinant antibodies as well? Our collection includes a mouse and a chimeric rabbit (original mouse ...

...for affecting change. On Friday, the symposium includes a panel discussion and keynote by former congressman...towards solutions. The 2015 Boston FoR meeting was designed to explore areas where further data is necessary...for students interested in leading the process despite the recent advance. Similarly, grad students throughout...development and implementation of new strategies designed to cope with current increases. These two rulings...

...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout... endjoining (NHEJ). Cas9-mediated NHEJ usually destroys the PAM site due its proximity to the cleavage...

...switches? FLEx (or “flip-excision”) switches were designed as a genetic tool for researchers to conditionally...does the FLEx switch work? Let’s say you want to design a genetic FLEx switch that turns BFP expression...single gene deleted in your knockout mouse, you can design a FLEx switch conditional knock-in cassette. As...experiment with a wildtype copy of a gene, you can also design FLEx switches to conditionally test the function...

...interest from its production site to its final destination. As an example, most secreted proteins are produced...pathway to their subcellular or extracellular destination. At the end of their life cycle, proteins may... about the subcellular structures your protein resides in and potentially which other proteins it forms...in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth review of quantitative colocalization...

... thanks to our guest blogger, Laura Desrochers! Laura Desrochers is currently a Postdoctoral Scientist...Apr 6, 2021. This post was contributed by Laura Desrochers, a postdoctoral scientist at AstraZeneca. Why...ve discovered.) As someone who has now seen both sides of the postdoc experience, I’m going to share what...